Pullulanase variants and polynucleotides encoding same

ABSTRACT

The present invention relates to pullulanase variants comprising substitutions of the parent pullulanase at one or more positions corresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/112,096filed Jul. 15, 2016, now allowed, which is a 35 U.S.C. 371 nationalapplication of PCT/EP2015/051145 filed Jan. 21, 2015, which claimspriority or the benefit under 35 U.S.C. 119 of European application nos.14152110.4 and 14195691.2 filed Jan. 22, 2014 and Dec. 1, 2014,respectively, the contents of which are fully incorporated herein byreference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to pullulanase variants, polynucleotidesencoding the variants, methods of producing the variants, and methods ofusing the variants. Also described is the use of pullulanase of theinvention for starch conversion to produce fermentation products. Theinvention also relates to a composition comprising a pullulanase of theinvention.

Description of the Related Art

Starch usually consists of about 80% amylopectin and 20% amylose.Amylopectin is a branched polysaccharide in which linear chainsalpha-1,4 D-glucose residues are joined by alpha-1,6 glucosidiclinkages. Amylopectin is partially degraded by alpha-amylase, whichhydrolyzes the 1,4-alpha-glucosidic linkages to produce branched andlinear oligosaccharides. Prolonged degradation of amylopectin byalpha-amylase results in the formation of so-called alpha-limit dextrinsthat are not susceptible to further hydrolysis by the alpha-amylase.Branched oligosaccharides can be hydrolyzed into linear oligosaccharidesby a debranching enzyme. The remaining branched oligosaccharides can bedepolymerized to D-glucose by glucoamylase, which hydrolyzes linearoligosaccharides into D-glucose.

Debranching enzymes which can attack amylopectin are divided into twoclasses: isoamylases (E.C. 3.2.1.68) and pullulanases (E.C. 3.2.1.41),respectively. Isoamylase hydrolyses alpha-1,6-D-glucosidic branchlinkages in amylopectin and beta-limit dextrins and can be distinguishedfrom pullulanases by the inability of isoamylase to attack pullulan, andby their limited action on alpha-limit dextrins.

It is well-known in the art to add isoamylases or pullulanases in starchconversion processes. Pullulanase is a starch debranching enzyme havingpullulan 6-glucano-hydrolase activity (EC3.2.1.41) that catalyzes thehydrolyses the α-1,6-glycosidic bonds in pullulan, releasing maltotriosewith reducing carbohydrate ends. Usually pullulanase is used incombination with an alpha amylase and/or a glucoamylase.

Pullulanases are known in the art. U.S. Pat. No. 6,074,854 and U.S. Pat.No. 5,817,498 disclose a pullulanase from Bacillus deramificans.WO2009/075682 disclose a pullulanase derived from Bacillusacidopullulyticus.

The present invention provides pullulanase variants with improvedproperties compared to its parent.

SUMMARY OF THE INVENTION

The present invention relates to a pullulanase variant comprising asubstitution at one or more positions corresponding to positions 393,143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430,456, 486, 492, 610, 631, 632, 665 and 699 of the polypeptide of SEQ IDNO: 3 wherein the variant has pullulanase activity; and a) wherein thevariant has at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3 and wherein the variant hasincreased thermoactivity compared to the pullulanase of SEQ ID NO: 3,particularly the variant has at least 60% relative activity whenmeasured at 70° C. relative to activity at 65° C., more particularly atleast 65%, more particularly at least 70%, more particularly at least60%, more particularly at least 75%, more particularly at least 80%,more particularly at least 90%, more particularly at least 100%; or b)wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or c) wherein the variant has at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 9 and wherein the variant has increased thermoactivitycompared to the pullulanase of SEQ ID NO: 9, particularly the varianthas at least 30% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 40%, more particularly atleast 50%, more particularly at least 60%, more particularly at least70%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%; or d) wherein the variant has at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 16 and wherein the variant has increasedthermoactivity compared to the pullulanase of SEQ ID NO: 16,particularly the variant has at least 30% relative activity whenmeasured at 70° C. relative to activity at 65° C., more particularly atleast 40%, more particularly at least 50%, more particularly at least60%, more particularly at least 70%, more particularly at least 80%,more particularly at least 90%, more particularly at least 100%; or e)wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In a further aspect the present invention relates to a variant catalyticdomain, wherein the variant catalytic domain comprises a substitution atone or more positions corresponding to positions 393, 346, 368, 370,373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 631, 632, 665 and699 of the polypeptide of SEQ ID NO: 3; and a) wherein the variantcatalytic domain has at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, but less than 100%sequence identity to amino acids 330 to 828 of SEQ ID NO: 3; or b)wherein the variant catalytic domain has at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; or c) wherein the variant catalytic domain has at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to amino acids 330 to829 of SEQ ID NO: 9; or d) wherein the variant has at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 16 and wherein the variant has increased thermoactivitycompared to the pullulanase of SEQ ID NO: 16, particularly the varianthas at least 30% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 40%, more particularly atleast 50%, more particularly at least 60%, more particularly at least70%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%; or e) wherein the variant has at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 17 and wherein the variant has increasedthermoactivity compared to the pullulanase of SEQ ID NO: 17,particularly the variant has at least 30% relative activity whenmeasured at 70° C. relative to activity at 65° C., more particularly atleast 40%, more particularly at least 50%, more particularly at least60%, more particularly at least 70%, more particularly at least 80%,more particularly at least 90%, more particularly at least 100%.

The present invention further relates to a pullulanase variantcomprising the variant catalytic domain of the invention, wherein thepullulanase variant has pullulanase activity and increasedthermoactivity compared to the parent pullulanase, and the variant hasat least 60% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 65%, more particularly atleast 70%, more particularly at least 60%, more particularly at least75%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%.

In a still further aspect the invention relates to compositionscomprising the pullulanase variants of the invention.

The present invention also relates to polynucleotides encoding thevariants; nucleic acid constructs, vectors, and host cells comprisingthe polynucleotides; and methods of producing the variants.

The present invention also relates to methods of producing afermentation product from a starch containing material.

Definitions

Pullulanase: The term “pullulanase” means a starch debranching enzymehaving pullulan 6-glucano-hydrolase activity (EC 3.2.1.41) thatcatalyzes the hydrolysis the α-1,6-glycosidic bonds in pullulan,releasing maltotriose with reducing carbohydrate ends. For purposes ofthe present invention, pullulanase activity can be determined accordingto the procedure described in the Examples. In the context of thepresent invention the variant pullulanases have improved thermoactivity.Increased thermoactivity was determined as relative activity whenmeasured at 70° C. relative to activity at 65° C. using the PHADEBASassay as described in the examples.

In particular the pullulanase variants of the invention have at least60% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 70%, more particularly at least 80%,more particularly at least 90%, more particularly at least 100% whencomparing variants of SEQ ID NO: 3.

In particular the pullulanase variants of the invention have at least30% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 40%, more particularly at least 50%,more particularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100% when comparing variants of SEQ ID NO: 6.

In particular the pullulanase variants of the invention have at least30% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 40%, more particularly at least 50%,more particularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100% when comparing variants of SEQ ID NO: 9.

Pullulanase domains, CBM41, X45, X25: According to NCBI's CDD (conserveddomain database, Marchler-Bauer et al., Nucleic acids research 2011,vol. 39, D225-229), X25, X45 and CBM41 domains in pullulanases aredescribed as follows:

X25 Domain of Bacillus acidopullulyticus Pullulanase and SimilarProteins.

Pullulanase (EC 3.2.1.41) cleaves 1.6-alpha-glucosidic linkages inpullulan, amylopectin, and glycogen, and in alpha- and beta-amylaselimit-dextrins of amylopectin and glycogen. Bacillus acidopullulyticuspullulanase is used industrially in the production of high fructose cornsyrup, high maltose content syrups and low calorie and “light” beers.Pullulanases, in addition to the catalytic domain, include severalcarbohydrate-binding domains (CBMs) as well as domains of unknownfunction (termed “X” modules). X25 was identified in Bacillusacidopullulyticus pullulanase, and splits another domain of unknownfunction (X45). X25 is present in multiple copies in some pullulanases.It has been suggested that X25 and X45 are CBMs which target mixedalpha-1.6/alpha-1.4 linked D-glucan polysaccharides.

X45 Domain of Bacillus acidopullulyticus Pullulanase and SimilarProteins.

Pullulanase (EC 3.2.1.41) cleaves 1.6-alpha-glucosidic linkages inpullulan, amylopectin, and glycogen, and in alpha- and beta-amylaselimit-dextrins of amylopectin and glycogen. Bacillus acidopullulyticuspullulanase is used industrially in the production of high fructose cornsyrup, high maltose content syrups and low calorie and “light” beers.Pullulanases, in addition to the catalytic domain, include severalcarbohydrate-binding domains (CBMs) as well as domains of unknownfunction (termed “X” modules). X45 was identified in Bacillusacidopullulyticus pullulanase; it is interrupted by another domain ofunknown function (X25). It has been suggested that X25 and X45 are CBMswhich target mixed alpha-1.6/alpha-1.4 linked D-glucan polysaccharides.

Family 41 Carbohydrate-Binding Module from Pullulanase-Like Enzymes

Pullulanases (EC 3.2.1.41) are a group of starch-debranching enzymes,catalyzing the hydrolysis of the alpha-1.6-glucosidic linkages ofalpha-glucans, preferentially pullulan. Pullulan is a polysaccharide inwhich alpha-1.4 linked maltotriosyl units are combined via an alpha-1.6linkage. These enzymes are of importance in the starch industry, wherethey are used to hydrolyze amylopectin starch. Pullulanases consist ofmultiple distinct domains, including a catalytic domain belonging to theglycoside hydrolase (GH) family 13 and carbohydrate-binding modules(CBM), including CBM41. Carbohydrate-Binding Module family 41, aremodules of approx. 100 residues found primarily in bacterialpullulanases. CBM41 alias PUD (Bacterial pullulanase-associated domain)modules, may be identified in a query protein sequence, by using thePfam database ‘Sequence Search’ tool available athttp://pfam.xfam.org/search, using Pfam version 26.0 or higher. The Pfamdatabase is a large collection of protein families, each represented bymultiple sequence alignments and hidden Markov models (HMMs). Pfam isfreely available under the Creative Commons Zero (“CC0”) license (seehttp://creativecommons.org/publicdomain/zero/1.0/).

The query protein sequence in FASTA format is entered into the searchfield of the Pfam database Sequence Search tool available via theinternet at http://pfam.xfam.org/search, and the Submit button ispressed, after which the Sequence Search results are displayed in atable showing Significant Pfam-A Matches, hereafter Table.

The presence of Table rows containing the Family name PUD are positiveidentifications of the presence of CBM41 alias PUD modules in the queryprotein sequence. The PUD Family name may also be referred to as PF03714without loss of ambiguity.

Additional columns in the Table show Envelope Start and End coordinates,which define respectively start and end coordinates of the CBM41 aliasPUD module in the query sequence, hereafter sequence Region whichencompasses sequence start to end inclusive.

An additional column in the Table shows E-value, which refers to thestatistical significance of the CBM41 alias PUD module identification.Lower E-values are statistically more significant than higher E-values.Significant CBM41 alias PUD module identifications are defined as thosehaving as having an E-value less than 1.0, preferably an E-value lessthan 1e-2 (0.01), more preferably an E-value less than 1e-4 (0.0001),even more preferably an E-value less than 1e-6 (0.000001).

Some pullulanases contain all these domains at their N-terminal and somelack either one or two or all of these domains.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a variant. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding avariant of the present invention. Each control sequence may be native(i.e., from the same gene) or foreign (i.e., from a different gene) tothe polynucleotide encoding the variant or native or foreign to eachother. Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a variant.

Expression: The term “expression” includes any step involved in theproduction of a variant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding a variantand is operably linked to control sequences that provide for itsexpression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids absent from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has pullulanaseactivity.

Hybrid pullulanase: Hybrid pullulanases according to the invention areobtained by combining an N-terminal fragment of one pullulanase fused toa C-terminal fragment of another pullulanase. Preferably the fusion istaking place within the catalytic domain where at least part of thecatalytic domain in the hybrid pullulanase should be derived from thecatalytic domain comprised in Promozyme D2 (SEQ ID NO: 5), however,other fusion points are also possible. The fusion could be a simplefusion between two fragments origination from the two parentpullulanases, however, the fusion could in some embodiments give rise toa shuffled amino acid sequence in the interface between the two parentfragments. Fusion should preferably be performed in a region homologybetween the parent pullulanases. The homologous region should at leastbe 4 amino acids.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Improved property: The term “improved property” means a characteristicassociated with a variant that is improved compared to the parent. Suchimproved properties include, but are not limited to, specific activity,thermo-stability and thermo-activity. In a particular embodiment theimproved property is increased thermoactivity. In another particularembodiment the improved property is increased thermostability. Inanother particular embodiment the improved property is increasedspecific activity.

Isolated: The term “isolated” means a substance in a form or environmentwhich does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Isoamylase: The term “isoamylase” means a starch debranching enzymeactivity (E.C. 3.2.1.68) that hydrolyses alpha-1,6-D-glucosidic branchlinkages in amylopectin and beta-limit dextrins and can be distinguishedfrom pullulanases by the inability of isoamylase to attack pullulan, andby the limited action on alpha-limit dextrins. Isoamylase may be addedin effective amounts well known to the person skilled in the art.Isoamylase may be added alone or together with a pullulanase.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 34 to 861 of SEQ ID NO: 2 and amino acids 1to 33 of SEQ ID NO: 2 are a signal peptide. In another aspect, themature polypeptide is amino acids 30 to 957 of SEQ ID NO: 5 and aminoacids 1 to 29 of SEQ ID NO: 5 are a signal peptide. In another aspect,the mature polypeptide is amino acids 34 to 862 of SEQ ID NO: 8 andamino acids 1 to 33 of SEQ ID NO: 8 are a signal peptide. It is known inthe art that a host cell may produce a mixture of two of more differentmature polypeptides (i.e., with a different C-terminal and/or N-terminalamino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving pullulanase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 100 to 2583 of SEQ ID NO: 1. Nucleotides1 to 99 of SEQ ID NO: 1 encode a signal peptide. In another aspect, themature polypeptide coding sequence is nucleotides 88 to 2871 of SEQ IDNO: 4. Nucleotides 1 to 87 of SEQ ID NO: 4 encode a signal peptide. Inanother aspect, the mature polypeptide coding sequence is nucleotides100 to 2586 of SEQ ID NO: 7. Nucleotides 1 to 99 of SEQ ID NO: 7 encodea signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Mutant: The term “mutant” means a polynucleotide encoding a variant.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Parent or parent pullulanase: The term “parent” or “parent pullulanaseor chimera pullulnase means a pullulanase to which an alteration is madeto produce the enzyme variants of the present invention. The parent maybe a naturally occurring (wild-type) polypeptide or a variant orfragment thereof.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the −nobrief option) is usedas the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having pullulanase activity.

Variant: The term “variant” means a polypeptide having pullulanaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition. The variants of the present invention have increasedthermo-activity compared to the parent enzyme.

In particular the pullulanase variants of the invention have at least60% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 70%, more particularly at least 80%,more particularly at least 90%, more particularly at least 100% whencomparing variants of SEQ ID NO: 3.

In particular the pullulanase variants of the invention have at least30% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 40%, more particularly at least 50%,more particularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100% when comparing variants of SEQ ID NO: 6.

In particular the pullulanase variants of the invention have at least30% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 40%, more particularly at least 50%,more particularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100% when comparing variants of SEQ ID NO: 9.

In particular the pullulanase variants of the invention have at least30% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 40%, more particularly at least 50%,more particularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100% when comparing variants of SEQ ID NO: 16.

In particular the pullulanase variants of the invention have at least30% relative activity when measured at 70° C. relative to activity at65° C., more particularly at least 40%, more particularly at least 50%,more particularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100% when comparing variants of SEQ ID NO: 17.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Wild-type Pullulanase: The term “wild-type” pullulanase means apullulanase expressed by a naturally occurring microorganism, such as abacterium, yeast, or filamentous fungus found in nature.

Conventions for Designation of Variants

For purposes of the present invention, the mature polypeptide comprisedin SEQ ID NO: 2 is used to determine the corresponding amino acidresidue in another pullulnase. In one embodiment, the mature polypeptideis disclosed as SED ID NO: 3. The amino acid sequence of anotherpullulanase is aligned with the mature polypeptide disclosed as SEQ IDNO: 3, and based on the alignment, the amino acid position numbercorresponding to any amino acid residue in the mature polypeptidedisclosed as SEQ ID NO: 3 is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276-277), preferably version 5.0.0 or later. Theparameters used are gap open penalty of 10, gap extension penalty of0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

Identification of the corresponding amino acid residue in anotherpullulanase can be determined by an alignment of multiple polypeptidesequences using several computer programs including, but not limited to,MUSCLE (multiple sequence comparison by log-expectation; version 3.5 orlater; Edgar, 2004, Nucleic Acids Research 32: 1792-1797), MAFFT(version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research30: 3059-3066; Katoh et al., 2005, Nucleic Acids Research 33: 511-518;Katoh and Toh, 2007, Bioinformatics 23: 372-374; Katoh et al., 2009,Methods in Molecular Biology 537: 39-64; Katoh and Toh, 2010,Bioinformatics 26: 1899-1900), and EMBOSS EMMA employing ClustalW (1.83or later; Thompson et al., 1994, Nucleic Acids Research 22: 4673-4680),using their respective default parameters.

When the other enzyme has diverged from the mature polypeptide of SEQ IDNO: 2 such that traditional sequence-based comparison fails to detecttheir relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), other pairwise sequence comparison algorithms can be used.Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations ofpolypeptide families (profiles) to search databases. For example, thePSI-BLAST program generates profiles through an iterative databasesearch process and is capable of detecting remote homologs (Atschul etal., 1997, Nucleic Acids Res. 25: 3389-3402). Even greater sensitivitycan be achieved if the family or superfamily for the polypeptide has oneor more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones, 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the polypeptide, and suchmodels can be assessed for accuracy using a variety of tools developedfor that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Two ormore protein structures can be aligned using a variety of algorithmssuch as the distance alignment matrix (Holm and Sander, 1998, Proteins33: 88-96) or combinatorial extension (Shindyalov and Bourne, 1998,Protein Engineering 11: 739-747), and implementation of these algorithmscan additionally be utilized to query structure databases with astructure of interest in order to discover possible structural homologs(e.g., Holm and Park, 2000, Bioinformatics 16: 566-567).

In describing the variants of the present invention, the nomenclaturedescribed below is adapted for ease of reference. The accepted IUPACsingle letter or three letter amino acid abbreviations are employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine at position 226 withalanine is designated as “Thr226Ala” or “T226A”. Multiple mutations areseparated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing substitutions at positions 205 and 411 ofglycine (G) with arginine (R) and serine (S) with phenylalanine (F),respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position, *. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, inserted aminoacid. Accordingly the insertion of lysine after glycine at position 195is designated “Gly195GlyLys” or “G195GK”. An insertion of multiple aminoacids is designated [Original amino acid, position, original amino acid,inserted amino acid #1, inserted amino acid #2; etc.]. For example, theinsertion of lysine and alanine after glycine at position 195 isindicated as “Gly195GlyLysAla” or “G195GKA”.

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample, the sequence would thus be:

Parent: Variant: 195 195 195a 195b G G - K - A

Multiple alterations. Variants comprising multiple alterations areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing a substitution of arginine and glycine atpositions 170 and 195 with tyrosine and glutamic acid, respectively.

Different alterations. Where different alterations can be introduced ata position, the different alterations are separated by a comma, e.g.,“Arg170Tyr,Glu” represents a substitution of arginine at position 170with tyrosine or glutamic acid. Thus, “Tyr167Gly,Ala+Arg170Gly,Ala”designates the following variants: “Tyr167Gly+Arg170Gly”,“Tyr167Gly+Arg170Ala”, “Tyr167Ala+Arg170Gly”, and “Tyr167Ala+Arg170Ala”.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to variant pullulanases derived from ahybrid parent pullulanase. The hybrid parent pullulanase is the subjectof the copending EP patent application 13176791.5 filed on Jul. 17,2013. The parent pullulanase, denoted P8, is a hybrid enzyme obtained bycombining an N-terminal fragment of the pullulanase derived fromBacillus acidopullullyticus described in WO 2009/075682 (SEQ ID NO: 4 inWO2009/075682; GENESEQP: AXB71624), fused to a C-terminal fragment of apullulanase derived from a Bacillus deramificans strain isolated from ahumus sample collected in Denmark (a homologous pullulanase fromBacillus deramificans was disclosed in U.S. Pat. No. 6,074,854 and U.S.Pat. No. 5,817,498). The resulting hybrid parent pullulanase isdisclosed herein as SEQ ID NO: 2, and the mature pullulanase as SEQ IDNO: 3.

The variants according to the present invention have improved propertiescompared to the parent. The improved properties are selected fromincreased thermo-activity or increased specific activity onmaltodextrin. In one embodiment the variants of the invention haveincreased thermoactivity. The positions to be substituted in order toobtain increased thermo-activity will be described in detail below. Inaddition to the effect observed when substituting any of the disclosedpositions in the parent pullulanase (the hybrid enzyme denoted as P8 orP008, and disclosed herein as SEQ ID NO: 3) some positions have alsobeen tested for alternative hybrids or wild type pullulanases. Two suchalternative hybrid pullulanases are disclosed herein as SEQ ID NO: 16and SEQ ID NO: 17. For more details on these specific constructs see theexample section herein. The results support the finding that the inparticular substitutions performed in the catalytic domain can beintroduced at the corresponding positions in other pullulanases, wildtype as well as hybrid, and the result will be variant pullulanaseshaving increased thermo-activity. When a specific substitution is testedin a different parent enzyme the starting amino acid present in theparent may be different from the first tested parent. This does not haveany effect on the resulting variant. As a general rule the essentialfeature is the amino acid that will be introduced at a specific positionnot what was there before the substitution. Thus throughout thespecification in many embodiments only the amino acid present aftersubstitution has been given. E.g., 393A means that whatever amino acidpresent in position 393 should be substituted to A (Ala).

The variants according to the invention comprises one or moresubstitutions numbered according to the mature polypeptide of SEQ ID NO:2, disclosed herein as SEQ ID NO: 3. Unless otherwise indicated theposition numbers referred to for variants disclosed herein refer tonumbering in SEQ ID NO: 3.

The present invention relates to pullulanase variant comprising asubstitution at one or more positions corresponding to positions 393,143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430,456, 486, 492, 610, 631, 632, 665 and 699 of the polypeptide of SEQ IDNO: 3 wherein the variant has pullulanase activity; and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Variants

The present invention provides pullulanase variants, comprising asubstitution at one or more positions corresponding to positions 393,143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402,429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 ofSEQ ID NO: 3 wherein the variant has pullulanase activity and increasedthermo-activity.

The variants may further comprise one or more additional alteration atone or more other positions. Such additional alterations may notsignificantly affect the properties of the variants according to theinvention but may change the % identity of the variant compared to SEQID NO: 3.

In an embodiment, the variant has sequence identity of at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100%, to the amino acidsequence of the parent pullulanase.

In another embodiment, the variant has at least at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, such as at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, sequence identity to the polypeptide of SEQ ID NO:3. In one aspect, the number of alterations in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5 such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 alterations.

In another embodiment, the variant has at least at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, such as at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, sequence identity to the polypeptide of SEQ ID NO:6. In one aspect, the number of alterations in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5 such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 alterations.

In another embodiment, the variant has at least at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, such as at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, sequence identity to the polypeptide of SEQ ID NO:9. In one aspect, the number of alterations in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5 such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 alterations.

In another embodiment, the variant has at least at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, such as at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, sequence identity to the polypeptide of SEQ ID NO:16. In one aspect, the number of alterations in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5 such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 alterations.

In another embodiment, the variant has at least at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, such as at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100%, sequence identity to the polypeptide of SEQ ID NO:17. In one aspect, the number of alterations in the variants of thepresent invention is 1-20, e.g., 1-10 and 1-5 such as 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 alterations.

In a particular embodiment the present invention relates to pullulanasevariant comprising a substitution at one or more positions correspondingto positions 393, 143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387,402, 429, 430, 456, 486, 492, 610, 631, 632, 665 and 699 of thepolypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity; and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, a variant comprises a substitution at two or morepositions corresponding to positions 393, 143, 150, 243, 244, 346, 368,370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 624, 631,632, 665 and 699 of the polypeptide of SEQ ID NO: 3 wherein the varianthas pullulanase activity; and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, a variant comprises a substitution at three or morepositions corresponding to positions 393, 143, 150, 243, 244, 346, 368,370, 373, 381, 385, 387, 402, 429, 430, 431, 456, 486, 492, 610, 624,631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3 wherein thevariant has pullulanase activity; and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, a variant comprises a substitution at four or morepositions corresponding to positions 393, 143, 150, 243, 244, 346, 368,370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610,624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3 whereinthe variant has pullulanase activity; and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, a variant comprises a substitution at five or morepositions corresponding to positions 393, 143, 150, 243, 244, 345, 346,368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492,610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3wherein the variant has pullulanase activity, and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, a variant comprises a substitution at six or morepositions corresponding to positions 393, 143, 150, 243, 244, 345, 346,368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486,492, 610, 624, 631, 632, 665 and 699.

In another aspect, a variant comprises a substitution at one positioncorresponding to positions 393, 143, 150, 243, 244, 346, 368, 456, 492,610, 624, or 699 of the polypeptide of SEQ ID NO: 3 wherein the varianthas pullulanase activity, and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 393. In another aspect, theamino acid at a position corresponding to position 393 is substitutedwith Ala, Arg, Asp, Cyt, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Ala. In another aspect,the variant comprise or consist of the substitution N393A of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 143. In another aspect, theamino acid at a position corresponding to position 143 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp or Tyr, preferably with Gly. In another aspect,the variant comprise or consist of the substitution V143G of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 150. In another aspect, theamino acid at a position corresponding to position 150 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Arg. In another aspect,the variant comprise or consist of the substitution E150R of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 243. In another aspect, theamino acid at a position corresponding to position 243 is substitutedwith Ala, Arg, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Glu. In another aspect,the variant comprise or consist of the substitution N243E of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 244. In another aspect, theamino acid at a position corresponding to position 244 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr or Val, preferably with Lys. In another aspect,the variant comprise or consist of the substitution S244K of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 345. In another aspect, theamino acid at a position corresponding to position 346 is substitutedwith Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Pro. In another aspect,the variant comprise or consist of the substitution A345P of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 346. In another aspect, theamino acid at a position corresponding to position 346 is substitutedwith Ala, Arg, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Ser. In another aspect,the variant comprise or consist of the substitution N346S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 368. In another aspect, theamino acid at a position corresponding to position 368 is substitutedwith Ala, Arg, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Glu. In another aspect,the variant comprise or consist of the substitution N368G of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 370. In another aspect, theamino acid at a position corresponding to position 370 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Ser. In another aspect,the variant comprise or consist of the substitution K370S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 373. In another aspect, theamino acid at a position corresponding to position 373 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Leu. In another aspect,the variant comprise or consist of the substitution I373L of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 381. In another aspect, theamino acid at a position corresponding to position 381 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Val. In another aspect,the variant comprise or consist of the substitution I381V of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 382. In another aspect, theamino acid at a position corresponding to position 382 is substitutedwith Ala, Arg, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Thr. In another aspect,the variant comprise or consist of the substitution N382T of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 385. In another aspect, theamino acid at a position corresponding to position 385 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Glu. In another aspect,the variant comprise or consist of the substitution Q385E of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 387. In another aspect, theamino acid at a position corresponding to position 387 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Leu. In another aspect,the variant comprise or consist of the substitution Q387L of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 402. In another aspect, theamino acid at a position corresponding to position 402 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Thr. In another aspect,the variant comprise or consist of the substitution M402T of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 429. In another aspect, theamino acid at a position corresponding to position 429 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Val. In another aspect,the variant comprise or consist of the substitution I429V of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 430. In another aspect, theamino acid at a position corresponding to position 430 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr or Val, preferably with Arg. In another aspect,the variant comprise or consist of the substitution T430R of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 431. In another aspect, theamino acid at a position corresponding to position 431 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Glutamic Acid. Inanother aspect, the variant comprise or consist of the substitutionQ431E of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 432. In another aspect, theamino acid at a position corresponding to position 432 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Phe. In another aspect,the variant comprise or consist of the substitution L432F of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 456. In another aspect, theamino acid at a position corresponding to position 456 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Ala. In another aspect,the variant comprise or consist of the substitution F456A of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 486. In another aspect, theamino acid at a position corresponding to position 486 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp or Tyr, preferably with Cys. In another aspect,the variant comprise or consist of the substitution V486C of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 492. In another aspect, theamino acid at a position corresponding to position 492 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr or Val, preferably with Ser or Ala. In anotheraspect, the variant comprise or consist of the substitution T492S,A ofthe polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 610. In another aspect, theamino acid at a position corresponding to position 610 is substitutedwith Ala, Arg, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Leu or Arginine. Inanother aspect, the variant comprise or consist of the substitutionN610L or N610R of the polypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 624. In another aspect, theamino acid at a position corresponding to position 624 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr or Val, preferably with Ser. In another aspect,the variant comprise or consist of the substitution G624S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 631. In another aspect, theamino acid at a position corresponding to position 631 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Trp, Tyr or Val, preferably with Ser. In another aspect,the variant comprise or consist of the substitution T631S of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 632. In another aspect, theamino acid at a position corresponding to position 632 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Thr, Trp, Tyr or Val, preferably with Cys. In another aspect,the variant comprise or consist of the substitution S632C of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 665. In another aspect, theamino acid at a position corresponding to position 665 is substitutedwith Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Ile, Leu, Lys, Met,Phe, Pro, Ser, Thr, Trp or Tyr, preferably with Ile. In another aspect,the variant comprise or consist of the substitution V665I of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of a substitutionat a position corresponding to position 699. In another aspect, theamino acid at a position corresponding to position 699 is substitutedwith Ala, Arg, Asn, Asp, Cys, Gin, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, Val, preferably with Arg. In another aspect,the variant comprise or consist of the substitution E699R of thepolypeptide of SEQ ID NO: 3.

In another aspect, the variant comprises or consists of one or moresubstitutions selected from the group consisting of 393A, 143G, 150R,243E, 244K, 345P, 346S, 368G, 370S, 373L, 381V, 382T, 385F, 387L, 402T,429V, 430R, 431E, 432F, 456A, 486C, 492S,A, 610R,L, 624S, 631S, 632C,665I and 699R, wherein position numbering refers to SEQ ID NO: 3.Throughout the present description in many occasions only the specificamino acids that should be present after the substitution is given. Thisis due to the fact that we have tested and found the substitutions canbe introduced in many different parent pullulanase and still result inthe same improved effect. The different parent pullulanases will not allhave the same amino acid in the same corresponding specific positionbefore substitution. Thus in order to obtain the described effect theessential feature is the specific amino acid present in a specificposition after substitution.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A, wherein the varianthas pullulanase activity; and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 432F, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 610R, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 624S, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions and 393A and 431E, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 432F, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 610R, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 624S, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 431E and 432F, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 431E and 610R, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 431E and 624S, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 432F and 610R, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 432F and 624S, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 610R and 624S, wherein the varianthas pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 432F, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 610R, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E and 432F, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E and 610R, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or d) wherein the variant has at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 16 and wherein the variant has increased thermoactivitycompared to the pullulanase of SEQ ID NO: 16, particularly the varianthas at least 30% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 40%, more particularly atleast 50%, more particularly at least 60%, more particularly at least70%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 432F and 610R, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 432F and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%;

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 610R and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 431E and 432F, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 431E and 610R, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 431E and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 432F and 610R, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 432F and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or e) wherein the variant has at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 17 and wherein the variant has increased thermoactivitycompared to the pullulanase of SEQ ID NO: 17, particularly the varianthas at least 30% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 40%, more particularly atleast 50%, more particularly at least 60%, more particularly at least70%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 610R and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 431E and 432F and 610R, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 431E and 432F and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 431E and 610R and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 432F and 610R and 624S, wherein thevariant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E and 432F,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E and 610R,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 432F and 610R,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 432F and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 610R and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E and 432F and 610R,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E and 432F and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E and 610R and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 432F and 610R and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 431E and 432F and 610R,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 431E and 432F and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 431E and 610R and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 432F and 610R and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 431E and 432F and 610R and 624S,wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E and 432F and610R, wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E and 432F and624S, wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E and 610R and624S,

wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 432F and 610R and624S, wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 431E and 432F and 610R and624S, wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 393A and 431E and 432F and 610R and624S, wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In another aspect, the variant comprises or consists of substitutions atpositions corresponding to positions 368G and 393A and 431E and 432F and610R and 624S, wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In a further particular embodiment the variant of the inventioncomprises at least one of the following substitutions or combinations ofspecific substitutions of the polypeptide of SEQ ID NO: 3 or in acorresponding position in the polypeptides of SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO: 16, SEQ ID NO: 17:

N368G; E699R; E150R; N346S; N243E; S244K; V143G; N393A; N610R; N610L;G624S; F456A; T492S,A; V486C+T492S,A; N368G+M402T; T631S+S632C;V486C+T492S,A+T631S+S632C; N393A+T631S+S632C; T631S+S632C+E699R;N393A+V486C+T492S,A+T631S+S632C; N393A+G624S+S632C;N393A+N610R+T631S+S632C; N393A+G624S+T631S+S632C;N393A+N610R+G624S+T631S+S632C; N393A+V486C+T492S,A+G624S+T631S+S632C;N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R;N346S+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+F456A+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;A345P+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N368G+K370S+I373L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;I381V+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;I381V+N382T+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;A345P+N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C+V665I;N393A+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+I429V+Q431E+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+I429V+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+A492S,A; N368G+N393A; N393A+N610R; N368G+N393A+N610R;N368G+N393A+T492S,A+N610R+G624S;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F;N368G+N393A+N610R+G624S+T631S+S632C;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C; orN368G+N393A+N610R+G624S+T631S+S632C+Q431E+L432F;

and wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

In a further particular embodiment the variant of the inventioncomprises at least one of the following substitutions or combinations ofspecific substitutions of the polypeptide of SEQ ID NO: 3 or in acorresponding position in the polypeptides of SEQ ID NO: 6, SEQ ID NO:9, SEQ ID NO: 16, SEQ ID NO: 17:

N368G; N393A; N610R; G624S; T492S,A;N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C; N368G+N393A+A492S,A;N368G+N393A+T492S,A+N610R+G624S;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F;N368G+N393A+N610R+G624S+T631S+S632C; N368G+N393A+N610R+G624S+T631S+S632C+Q431E+L432F;

and wherein the variant has pullulanase activity, and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

d) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or

e) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N368G of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution E699R of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution E150R of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N346S of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N243E of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution S244K of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution V143G of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N393A of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N610R of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N610L of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution G624S of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution F456A of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution T492S,A of the polypeptide of SEQID NO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution V486C+T492S,A of the polypeptideof SEQ ID NO: 3, wherein the variant has pullulanase activity, andwherein the pullulanase variants have at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N368G+M402T of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution T631S+S632C of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution V486C+T492S,A+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N393A+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution T631S+S632C+E699R of thepolypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N393A+V486C+T492S,A+T631S+S632C ofthe polypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N393A+G624S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N393A+N610R+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N393A+G624S+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitution N393A+N610R+G624S+T631S+S632C ofthe polypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+V486C+T492S,A+G624S+T631S+S632C of the polypeptide of SEQ ID NO:3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+V486C+T492S,A+N610R+G624S+T631S+S632C of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN368G+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN346S+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+F456A+V486C+T492S,A+N610R+G624S+T631S+S632C of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+T492S,A+N610R+G624S+T631S+S632C of the polypeptide of SEQ ID NO:3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN368G+N393A+T492S,A+N610R+G624S+T631S+S632C of the polypeptide of SEQ IDNO: 3, wherein the variant has pullulanase activity, and wherein thepullulanase variants have at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionA345P+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN368G+K370S+I373L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionI381V+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionI381V+N382T+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C ofthe polypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionA345P+N368G+N393A+T492S,A+N610R+G624S+T631S+S632C of the polypeptide ofSEQ ID NO: 3, wherein the variant has pullulanase activity, and whereinthe pullulanase variants have at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionA345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C ofthe polypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionA345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C+V665Iof the polypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+I429V+Q431E+V486C+T492S,A+N610R+G624S+T631S+S632C of thepolypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

More specifically the invention relates in one embodiment to pullulanasevariants, comprising the substitutionN393A+I429V+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C ofthe polypeptide of SEQ ID NO: 3, wherein the variant has pullulanaseactivity, and wherein the pullulanase variants have at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 3.

All of the above specific substitutions or combinations of substitutionshave an effect of increased thermoactivity of the resulting variant whenintroduced in the parent disclosed as SEQ ID NO: 3. In order to test theeffect of the specific substitutions in a different parent enzyme,several substitutions and combination of substitutions have beenintroduced in the parent enzyme disclosed herein as SEQ ID NO: 6. Allthe tested substitutions were shown also to be effective in SEQ ID NO:6. SEQ ID NO: 6 has an additional 100 amino acids compared to SEQ ID NO:3. Thus the actual position numbers are +100. The additional 100 aminoacids constitute what is known as an X25 domain (domain of unknownfunction) of 104 amino acids and deletions corresponding to 4 aminoacids in the N-terminal part. The X25 domain is found in the N-terminalpart of SEQ ID NO: 6 in positions 158-261, upstream of the catalyticdomain.

In a particular embodiment the variant of the invention comprises atleast one of the following substitutions or combinations of specificsubstitutions of the polypeptide of SEQ ID NO: 3 or in a correspondingposition in the polypeptides of SEQ ID NO: 6:

N368G; N393A; N610R; G624S; N368G+N393A+A492S,A; N368G+N393A;N393A+N610R; N368G+N393A+N610R;

N368G+N393A+T492S,A+N610R+G624S; and wherein the variant has pullulanaseactivity, and wherein the variant has at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6and wherein the variant has increased thermoactivity compared to thepullulanase of SEQ ID NO: 6, particularly the variant has at least 30%relative activity when measured at 70° C. relative to activity at 65°C., more particularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Similarly, in a further embodiments specific combinations ofsubstitutions were tested in parent hybrid pullulanases disclosed hereinas SEQ ID NO: 16 and SEQ ID NO: 17.

Thus in a particular embodiment the variant of the invention comprisesat least one of the following substitutions or combinations of specificsubstitutions of the polypeptide of SEQ ID NO: 3 or in a correspondingposition in the polypeptides of SEQ ID NO: 16 or SEQ ID NO: 17:

N368G+N393A+N610R+G624S+T631 S+S632C;N368G+N393A+N610R+G624S+T631S+S632C+Q431E+L432F;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C; orN368G+N393A+T492S,A+N610R+G624S+T631 S+S632C+Q431E+L432F;

and wherein the variant has pullulanase activity, and wherein thevariant has at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to the polypeptide of SEQ ID NO: 16 or SEQ ID NO: 17 andwherein the variant has increased thermoactivity compared to thepullulanase of SEQ ID NO: 16 or SEQ ID NO: 17, particularly the varianthas at least 30% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 40%, more particularly atleast 50%, more particularly at least 60%, more particularly at least70%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%.

When the parent pullulanase is selected as SEQ ID NO: 6, SEQ ID NO: 16,or SEQ ID NO: 17, which have 100 amino acids more than e.g., SEQ ID NO:3 the actual positions referring to SEQ ID NO: 6 SEQ ID NO: 16, or SEQID NO: 17, numbering will be +100. Thus as an example,N368G+N393A+A492S, would correspond to N468G+N493A+A592S in SEQ ID NO:6.

Variant Catalytic Domain

The present invention provides a variant catalytic domain, wherein thevariant catalytic domain comprises a substitution at one or morepositions corresponding to positions 393, 346, 368, 370, 373, 381, 385,387, 402, 429, 430, 456, 486, 492, 610, 631, 632, 665 and 699 of thepolypeptide of SEQ ID NO: 3; and

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 828 of SEQ IDNO: 3; orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

In another aspect, a variant catalytic domain comprises a substitutionat two or more positions corresponding to positions 393, 346, 368, 370,373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 624, 631, 632,665 and 699 of the polypeptide of SEQ ID NO: 3; and

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 828 of SEQ IDNO: 3; orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

In another aspect, a variant catalytic domain comprises a substitutionat three or more positions corresponding to positions 393, 346, 368,370, 373, 381, 385, 387, 402, 429, 430, 431, 456, 486, 492, 610, 624,631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3; and

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 828 of SEQ IDNO: 3; orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

In another aspect, a variant catalytic domain comprises a four or morepositions corresponding to positions 393, 346, 368, 370, 373, 381, 385,387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and699 of the polypeptide of SEQ ID NO: 3; and

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 828 of SEQ IDNO: 3; orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

In another aspect, a variant catalytic domain comprises a five or morepositions corresponding to positions 393, 345, 346, 368, 370, 373, 381,385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632,665 and 699 of the polypeptide of SEQ ID NO: 3; and

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 828 of SEQ IDNO: 3; or

b) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; or

c) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; or

d) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; or

e) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

In another aspect, a variant catalytic domain comprises a substitutionat six or more positions corresponding to positions 393, 345, 346, 368,370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492,610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3; and

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 828 of SEQ IDNO: 3; orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

In a further embodiment the present invention relates to a pullulanasevariant comprising the variant catalytic domain according to theinvention, wherein the pullulanase variant has pullulanase activity andincreased thermoactivity compared to the parent pullulanase, and thevariant has at least 60% relative activity when measured at 70° C.relative to activity at 65° C., more particularly at least 65%, moreparticularly at least 70%, more particularly at least 60%, moreparticularly at least 75%, more particularly at least 80%, moreparticularly at least 90%, more particularly at least 100%.

The pullulanase variant comprising the variant catalytic domain,comprises one or more substitutions selected from the group consistingof 393A, 345P, 346S, 368G, 370S, 373L, 381V, 382T, 385F, 387L, 402T,429V, 430R, 431E, 432F, 456A, 486C, 492S,A, 610R,L, 624S, 631S, 632C,665I and 699R corresponding to positions of polypeptide of SEQ ID NO: 3.

According to the invention several variant catalytic domains (CD) havebeen constructed and it has further been shown in the examples that theresulting variant pullulanases having increased thermo-activity can befurther modified by replacing the N-terminal part of the maturepullulanase and at the same time retain the improved properties, e.g.,increased thermo-activity.

Pullulanases may comprise N-terminal parts comprising domains selectedfrom a CBM41 domain, an X45 domain and a CBM48 domain. Thus in oneembodiment the pullulanase variant according to the invention, furthercomprises an N-terminal part comprising at least one of the domainsselected from a CBM41 domain, an X45 domain and a CBM48 domain. In oneembodiment all of these domains may be present.

The pullulanase variant according to the invention may further comprisean X25 domain. Preferably the N-terminal domains are selected fromCBM41, X45, X25, and CBM48 obtainable from a Bacillus bacterium.Particularly the Bacillus species is Bacillus acidopullulyticus orBacillus deramificans.

In a particular embodiment the pullulanase variant according to theinvention comprises or consists of one or more substitutions selectedfrom the group consisting of 393A, 368G, 492S,A, 610R,L, 624S, 631S,632C, 431E, 432F. More particularly the variant catalytic domaincomprises at least one of the following substitutions or combinations ofsubstitutions:

368G+393A+492S,A; 368G+393A+T492A,S+610R+624S; 393A+492S,A+610R+624S+631S+632C; 368G+393A+492S,A+610R+624S+631 S+632C;368G+393A+492S,A+610R+624S+631 S+632C+431E+432F;368G+393A+610R+624S+631S+632C; or368G+393A+610R+624S+631S+632C+431E+432F;

anda) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 828 of SEQ IDNO: 3; orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 6; orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

Particular examples of variants according to the invention, in whichN-terminal part have been replaced are further described in theexamples. Thus in further particular embodiments the invention relatesto pullulanase variant selected from SEQ ID NO: 20 or a pullulanasehaving at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99%, or 100% sequence identity to thepolypeptide of SEQ ID NO: 20, and comprising the substitutionsN368G+N393A+N610R+G624S+T631S+S632C, using SEQ ID NO: 3 for numbering.In another particular embodiment the invention relates to pullulanasevariant selected from SEQ ID NO: 21 or a pullulanase having at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 21, and comprising the substitutions N368G+N393A+A492S,A,using SEQ ID NO: 3 for numbering. In another particular embodiment theinvention relates to pullulanase variant selected from SEQ ID NO: 22 ora pullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 22, and comprising thesubstitutions N368G+N393A+T492S,A+N610R+G624S, using SEQ ID NO: 3 fornumbering. In another particular embodiment the invention relates topullulanase variant selected from SEQ ID NO: 23 or a pullulanase havingat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, or 100% sequence identity to the polypeptideof SEQ ID NO: 23, and comprising the substitutionsN368G+N393A+T492S,A+N610R+G624S, using SEQ ID NO: 3 for numbering. Inanother particular embodiment the invention relates to pullulanasevariant selected from SEQ ID NO: 24 or a pullulanase having at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 24, and comprising the substitutionsN368G+N393A+T492S,A+N610R+G624S+T631S+S632C, using SEQ ID NO: 3 fornumbering. In another particular embodiment the invention relates topullulanase variant selected from SEQ ID NO: 25 or a pullulanase havingat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, or 100% sequence identity to the polypeptideof SEQ ID NO: 25, and comprising the substitutionsN368G+N393A+T492S,A+N610R+G624S+T631S+S632C, using SEQ ID NO: 3 fornumbering. In another particular embodiment the invention relates topullulanase variant selected from SEQ ID NO: 26 or a pullulanase havingat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, or 100% sequence identity to the polypeptideof SEQ ID NO: 26, and comprising the substitutionsN368G+N393A+N610R+G624S+T631S+S632C+Q431E+L432F, using SEQ ID NO: 3 fornumbering. In another particular embodiment the invention relates topullulanase variant selected from SEQ ID NO: 27 or a pullulanase havingat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, or 100% sequence identity to the polypeptideof SEQ ID NO: 27, and comprising the substitutionsN368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F, using SEQ IDNO: 3 for numbering. In another particular embodiment the inventionrelates to pullulanase variant selected from SEQ ID NO: 28 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 28, and comprising thesubstitutions N368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F,using SEQ ID NO: 3 for numbering.

In a further aspect of the invention thermoactivity may be furtherimproved by the addition of further specific substitutions (using SEQ IDNO: 6 for numbering) selected from one of the following substitutions orcombinations of substitutions:

Q258A; Q287R; Q352A; Q356R; Q258A+Q352A+Q356R; Q258A+Q287R+Q352A+Q356R;V212I; H186A; V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+V212I+Q258A+Q287R+Q352A+Q356R; Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q485E+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+Q487L+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+S557A+L559G+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+H421E+Q487L+S557A+L559G+V586A+D686S+E799R.

In a particular embodiment the invention relates to pullulanase variantsselected from a pullulanase having at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the polypeptide of SEQ ID NO: 26, and comprising thesubstitutions (using SEQ ID NO: 6 for numbering)N468G+N493A+N710R+G724S+T731S+S732C+Q531E+L532F, and wherein thevariants further comprises one of the following substitutions orcombinations of substitutions:

Q258A; Q287R; Q352A; Q356R; Q258A+Q352A+Q356R; Q258A+Q287R+Q352A+Q356R;V212I; H186A; V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+V212I+Q258A+Q287R+Q352A+Q356R; Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q485E+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+Q487L+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+S557A+L559G+D686S+E799R;

N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+H421E+Q487L+S557A+L559G+V586A+D686S+E799R;said variants having at least 60% relative activity when measured at 72°C. relative to activity at 65° C. using the PHADEBAS assay.

In a further particular embodiment the invention relates to pullulanasevariants selected from a pullulanase having at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the polypeptide of SEQ ID NO: 27, and comprisingthe substitutions (using SEQ ID NO: 6 for numbering)N468G+N493A+T592S,A+N710R+G724S+T731S+S732C+Q531E+L532F, and wherein thevariants further comprises one of the following substitutions orcombinations of substitutions:

Q258A; Q287R; Q352A; Q356R; Q258A+Q352A+Q356R; Q258A+Q287R+Q352A+Q356R;V212I; H186A; V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+V212I+Q258A+Q287R+Q352A+Q356R; Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q485E+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+Q487L+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+S557A+L559G+D686S+E799R;

N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+H421E+Q487L+S557A+L559G+V586A+D686S+E799R;said variants having at least 60% relative activity when measured at 72°C. relative to activity at 65° C. using the PHADEBAS assay.

In another aspect of the invention thermoactivity may be furtherimproved by the addition of further specific substitutions (using SEQ IDNO: 3 for numbering) selected from one of the following substitutions orcombinations of substitutions:

Y27K+H79Y+Q187R+S798R; Y27K+H79Y+Q187R+D586S+S798R;Y27K+H79Y+Q187R+D586S+E699R+S798R; Y27K+H79Y+Q187R+T486S+D586S+S798R;N19G+Y27K+H79Y+Q187R+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q385E+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+Q459G+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+H321E+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+Q459G+D586S+E699R+S798R.

In a particular embodiment the invention relates to pullulanase variantsselected from a pullulanase having at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the polypeptide of SEQ ID NO: 28, and comprising thesubstitutions (using SEQ ID NO: 3 for numbering)N368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F, and wherein thevariants further comprises one of the following substitutions orcombinations of substitutions:

Y27K+H79Y+Q187R+S798R; Y27K+H79Y+Q187R+D586S+S798R;Y27K+H79Y+Q187R+D586S+E699R+S798R; Y27K+H79Y+Q187R+T486S+D586S+S798R;N19G+Y27K+H79Y+Q187R+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q385E+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+Q459G+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+H321E+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+D586S+E699R+S798R;

N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+Q459G+D586S+E699R+S798R, saidvariants having at least 30% relative activity when measured at 76° C.relative to activity at 65° C. using the PHADEBAS assay.

The variants may further comprise one or more additional substitutionsat one or more (e.g., several) other positions. Such further variationcould be introduced without affecting significantly the properties ofthe pullulanase variants.

The amino acid changes may be of a minor nature, that is conservativeamino acid substitutions or insertions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof 1-30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up to20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R.L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for pullulanase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Parent Pullulanase

The parent pullulanase, may be a hybrid enzyme, e.g., P008 (SEQ ID NO:3), as described herein obtained by combining an N-terminal fragment ofthe pullulanase derived from Bacillus acidopullulyticus described in WO2009/075682 (SEQ ID NO: 4 in WO2009/075682; GENESEQP: AXB71624), fusedto a C-terminal fragment of a pullulanase derived from a Bacillusderamificans strain isolated from a humus sample collected in Denmark (ahomologous pullulanase from Bacillus deramificans was disclosed in U.S.Pat. No. 6,074,854 and U.S. Pat. No. 5,817,498). Further parentpullulanases disclosed herein include P258, disclosed herein as SEQ IDNO: 16 or P243, disclosed herein as SEQ ID NO: 17, P006 disclosed hereinas SEQ ID NO: 19.

The parent pullulanase may also be any wildtype, or variant or hybridpullulanase that may advantageously be improved by increasing thethermo-activity by introducing one or more substitutions as specifiedherein.

The parent pullulanase may be (a) a polypeptide having at least 70%sequence identity to the mature polypeptide of SEQ ID NO: 2 or 5 or 8;(b) a polypeptide encoded by a polynucleotide that hybridizes under lowstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1 or 4 or 7, (ii) the full-length complement of (i) or (c) apolypeptide encoded by a polynucleotide having at least 60% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 or 4or 7.

In an aspect, the parent has a sequence identity to the maturepolypeptide of SEQ ID NO: 2 or 5 or 8 of at least 75%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have pullulanase activity. In one aspect, theamino acid sequence of the parent differs by up to 10 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the mature polypeptide of SEQ IDNO: 2 or 5 or 8.

In another aspect, the parent comprises or consists of the amino acidsequence of SEQ ID NO: 2 or 5 or 8. In another aspect, the parentcomprises or consists of the mature polypeptide of SEQ ID NO: 2 or 5 or8. In another aspect, the parent comprises or consists of amino acids 34to 861 of SEQ ID NO: 2; 30 to 957 of SEQ ID NO: 5 or 34 to 862 of SEQ IDNO: 8 respectively.

In another embodiment, the parent is an allelic variant of the maturepolypeptide of SEQ ID NO: 2 or 5 or 8.

In another aspect, the parent is encoded by a polynucleotide thathybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1 or 4 or 7, (ii) the full-length complement of (i) (Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold SpringHarbor, N.Y.).

The polynucleotide of SEQ ID NO: 1 or 4 or 7 and their subsequencethereof, as well as the polypeptide of SEQ ID NO: 2 or 5 or 8 and theirfragment thereof, may be used to design nucleic acid probes to identifyand clone DNA encoding a parent from strains of different genera orspecies according to methods well known in the art. In particular, suchprobes can be used for hybridization with the genomic DNA or cDNA of acell of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 15, e.g., at least 25, at least 35, or at least 70nucleotides in length. Preferably, the nucleic acid probe is at least100 nucleotides in length, e.g., at least 200 nucleotides, at least 300nucleotides, at least 400 nucleotides, at least 500 nucleotides, atleast 600 nucleotides, at least 700 nucleotides, at least 800nucleotides, or at least 900 nucleotides in length. Both DNA and RNAprobes can be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a parent. Genomic or other DNA from such other strains may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA that hybridizeswith SEQ ID NO: 1 or 4 or 7 or a subsequence thereof, the carriermaterial is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1 or 4 or 7; (ii) the mature polypeptide codingsequence of SEQ ID NO: 1 or 4 or 7; (iii) the full-length complementthereof; or (iv) a subsequence thereof; under very low to very highstringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using, for example,X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1, 4 or 7. In another aspect, the nucleic acidprobe is nucleotides 100 to 2583 of SEQ ID NO: 1; nucleotides 88 to 2871of SEQ ID NO: 4 or nucleotides 100 to 2586 of SEQ ID NO: 7. In anotheraspect, the nucleic acid probe is a polynucleotide that encodes thepolypeptide of SEQ ID NO: 2, 5 or 8; the mature polypeptide thereof; ora fragment thereof. In another aspect, the nucleic acid probe is SEQ IDNO: 1, 4, or 7.

In another embodiment, the parent is encoded by a polynucleotide havinga sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 60%, e.g., at least 65%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The parent may be a fusion polypeptide or cleavable fusion polypeptidein which another polypeptide is fused at the N-terminus or theC-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The parent may be obtained from microorganisms of any genus. Forpurposes of the present invention, the term “obtained from” as usedherein in connection with a given source shall mean that the parentencoded by a polynucleotide is produced by the source or by a strain inwhich the polynucleotide from the source has been inserted. In oneaspect, the parent is secreted extracellularly.

The parent may be a bacterial pullulanase for example, the parent may bea Gram-positive bacterial polypeptide such as a Bacillus.

In one aspect, the parent is a Bacillus acidopullulyticus, or Bacillusderamificans pullulanase.

In another aspect, the parent is a Bacillus pullulanase, e.g., thepullulanase of SEQ ID NO: 2 or 5 or 8 or the mature polypeptide thereof.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent may be identified and obtained from other sources includingmicroorganisms isolated from nature (e.g., soil, composts, water, etc.)or DNA samples obtained directly from natural materials (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms and DNA directly from natural habitats are wellknown in the art. A polynucleotide encoding a parent may then beobtained by similarly screening a genomic DNA or cDNA library of anothermicroorganism or mixed DNA sample. Once a polynucleotide encoding aparent has been detected with the probe(s), the polynucleotide can beisolated or cloned by utilizing techniques that are known to those ofordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Preparation of Variants

The present invention also relates to methods for obtaining a varianthaving pullulanase activity, comprising: (a) introducing into a parentpullulanase a substitution at one or more (e.g., several) positionscorresponding to positions 393, 143, 150, 243, 244, 345, 346, 368, 370,373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486, 492, 610,624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3, 6, 9 orhybrids thereof, wherein the variant has pullulanase activity; and (b)recovering the variant.

The variants can be prepared using any mutagenesis procedure known inthe art, such as site-directed mutagenesis, synthetic gene construction,semi-synthetic gene construction, random mutagenesis, shuffling, etc.

Site-directed mutagenesis is a technique in which one or more (e.g.,several) mutations are introduced at one or more defined sites in apolynucleotide encoding the parent.

Site-directed mutagenesis can be accomplished in vitro by PCR involvingthe use of oligonucleotide primers containing the desired mutation.Site-directed mutagenesis can also be performed in vitro by cassettemutagenesis involving the cleavage by a restriction enzyme at a site inthe plasmid comprising a polynucleotide encoding the parent andsubsequent ligation of an oligonucleotide containing the mutation in thepolynucleotide. Usually the restriction enzyme that digests the plasmidand the oligonucleotide is the same, permitting sticky ends of theplasmid and the insert to ligate to one another. See, e.g., Scherer andDavis, 1979, Proc. Natl. Acad. Sci. USA 76: 4949-4955; and Barton etal., 1990, Nucleic Acids Res. 18: 7349-4966.

Site-directed mutagenesis can also be accomplished in vivo by methodsknown in the art. See, e.g., U.S. Patent Application Publication No.2004/0171154; Storici et al., 2001, Nature Biotechnol. 19: 773-776; Krenet al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996,Fungal Genet. Newslett. 43: 15-16.

Any site-directed mutagenesis procedure can be used in the presentinvention. There are many commercial kits available that can be used toprepare variants.

Synthetic gene construction entails in vitro synthesis of a designedpolynucleotide molecule to encode a polypeptide of interest. Genesynthesis can be performed utilizing a number of techniques, such as themultiplex microchip-based technology described by Tian et al. (2004,Nature 432: 1050-1054) and similar technologies wherein oligonucleotidesare synthesized and assembled upon photo-programmable microfluidicchips.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

Semi-synthetic gene construction is accomplished by combining aspects ofsynthetic gene construction, and/or site-directed mutagenesis, and/orrandom mutagenesis, and/or shuffling. Semi-synthetic construction istypified by a process utilizing polynucleotide fragments that aresynthesized, in combination with PCR techniques. Defined regions ofgenes may thus be synthesized de novo, while other regions may beamplified using site-specific mutagenic primers, while yet other regionsmay be subjected to error-prone PCR or non-error prone PCRamplification. Polynucleotide subsequences may then be shuffled.

Polynucleotides

The present invention also relates to polynucleotides encoding a variantof the present invention.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of a variant. Manipulation of the polynucleotide prior toits insertion into a vector may be desirable or necessary depending onthe expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide which isrecognized by a host cell for expression of the polynucleotide. Thepromoter contains transcriptional control sequences that mediate theexpression of the variant. The promoter may be any polynucleotide thatshows transcriptional activity in the host cell including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIll, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof. Other promoters are describedin U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminatorsequence is operably linked to the 3′-terminus of the polynucleotideencoding the variant. Any terminator that is functional in the host cellmay be used.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseV, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, andTrichoderma reesei translation elongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIA gene (WO 94/25612) and a Bacillus subtilisSP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leadersequence is operably linked to the 5′-terminus of the polynucleotideencoding the variant. Any leader that is functional in the host cell maybe used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the variant-encoding sequence and,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a variant anddirects the variant into the cell's secretory pathway. The 5′-end of thecoding sequence of the polynucleotide may inherently contain a signalpeptide coding sequence naturally linked in translation reading framewith the segment of the coding sequence that encodes the variant.Alternatively, the 5′-end of the coding sequence may contain a signalpeptide coding sequence that is foreign to the coding sequence. Aforeign signal peptide coding sequence may be required where the codingsequence does not naturally contain a signal peptide coding sequence.Alternatively, a foreign signal peptide coding sequence may simplyreplace the natural signal peptide coding sequence in order to enhancesecretion of the variant. However, any signal peptide coding sequencethat directs the expressed variant into the secretory pathway of a hostcell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a variant. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of the variantand the signal peptide sequence is positioned next to the N-terminus ofthe propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the variant relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the variant would be operably linkedwith the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide encoding a variant of the present invention,a promoter, and transcriptional and translational stop signals. Thevarious nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the variant at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the variant or any other element ofthe vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a variant. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide encoding a variant of the present invention operablylinked to one or more control sequences that direct the production of avariant of the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thevariant and its source.

The host cell may be any cell useful in the recombinant production of avariant, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausi, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell, including,but not limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397), or conjugation (see,e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or conjugation (see,e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any methodknown in the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates method for producing a variantpullulanase of a parent pullulanase comprising substitution of theparent pullulanase at one or more positions corresponding to positions393, 143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387,402, 429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699of the polypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity and increased thermoactivity compared to the parent; and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3; or

b) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6; or

c) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9.

A further embodiment relates to variant pullulanases produced by themethod of the invention.

In another aspect, method of producing a variant, comprising: (a)cultivating a host cell of the present invention under conditionssuitable for expression of the variant; and (b) recovering the variant.

The host cells are cultivated in a nutrient medium suitable forproduction of the variant using methods known in the art. For example,the cell may be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing the variantto be expressed and/or isolated. The cultivation takes place in asuitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the variant is secreted into the nutrient medium, thevariant can be recovered directly from the medium. If the variant is notsecreted, it can be recovered from cell lysates.

The variant may be detected using methods known in the art that arespecific for the variants. These detection methods include, but are notlimited to, use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the variant.

The variant may be recovered using methods known in the art. Forexample, the variant may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The variant may be purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure variants.

In an alternative aspect, the variant is not recovered, but rather ahost cell of the present invention expressing the variant is used as asource of the variant.

Fermentation Broth Formulations or Cell Compositions

The present invention also relates to a fermentation broth formulationor a cell composition comprising a polypeptide of the present invention.The fermentation broth product further comprises additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. In some embodiments, the composition is a cell-killed wholebroth containing organic acid(s), killed cells and/or cell debris, andculture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compositions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis. In some embodiments, the cell-killed whole broth orcomposition contains the spent cell culture medium, extracellularenzymes, and killed filamentous fungal cells. In some embodiments, themicrobial cells present in the cell-killed whole broth or compositioncan be permeabilized and/or lysed using methods known in the art. Awhole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Enzyme Compositions

The present invention also relates to compositions comprising apullulanase variant of the present invention. Preferably, thecompositions are enriched in such a polypeptide. The term “enriched”indicates that the pullulanase activity of the composition has beenincreased, e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as the pullulanase variant according to the inventionand one or more (e.g., several) enzymes selected from the groupconsisting of hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, alpha-amylase, beta-amylase, beta-galactosidase,beta-glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, glucoamylase, invertase, laccase, lipase, mannosidase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase,polyphenoloxidase, protease, ribonuclease, transglutaminase, orxylanase. Preferably the enzyme activities comprised in the compositionare selected from the hybrid pullulanase according to the invention andone or more enzymes selected from the group consisting of glucoamylase,alpha-amylase, beta-amylase, and protease. In one particular embodimentthe composition comprises a pullulanase, a glucoamylase, analpha-amylase and a protease. In another particular embodiment thecomposition comprises a pullulanase, an alpha-amylase and a protease. Inanother particular embodiment the composition comprises a pullulanase, aglucoamylase, and an alpha-amylase. In another particular embodiment thecomposition comprises a pullulanase, and a beta-amylase.

In a particular embodiment the composition comprises the variantpullulanase of the invention and an alpha amylase. Preferred arebacterial alpha-amylases, which typically are stable at temperaturesused during liquefaction. In a preferred embodiment the alpha-amylase isderived from Bacillus stearothermophilus. The Bacillusstearothermophilus alpha-amylase may be a mature wild-type or a maturevariant thereof. The mature Bacillus stearothermophilus alpha-amylasesmay naturally be truncated during recombinant production. For instance,the Bacillus stearothermophilus alpha-amylase may be truncated so it hasaround 491 amino acids compared to SEQ ID NO: 3 in WO 99/19467.Preferred are Bacillus alpha-amylases, especially Bacillusstearothermophilus alpha-amylases, which have a double deletioncorresponding to a deletion of positions 181 and 182 and furthercomprise a N193F substitution (also denoted I181*+G182*+N193F) comparedto the wild-type BSG alpha-amylase amino acid sequence set forth in SEQID NO: 3 disclosed in WO 99/19467. The bacterial alpha-amylase may alsohave a substitution in a position corresponding to S239 in the Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, or aS242 variant of the Bacillus stearothermophilus alpha-amylase of SEQ IDNO: 3 in WO 99/19467. In a preferred embodiment the alpha-amylase isselected from the group of Bacillus stearomthermphilus alpha-amylasevariants:

I181*+G182*+N193F+E129V+K177L+R179E;I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; andI181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQID NO: 3 disclosed in WO 99/19467 for numbering).

In another preferred embodiment the alpha-amylase is an alpha-amylasederived from Rhizomucor pusillus with an Aspergillus niger glucoamylaselinker and starch-binding domain (SBD), preferably the one shown in SEQID NO: 7 in WO2013/006756, preferably having one or more of thefollowing substitutions: G128D, D143N, especially G128D+D143N.

In another particular embodiment the composition comprises the variantpullulanase of the invention, and a protease. In an preferred embodimentthe protease is a variant of the Thermoascus aurantiacus metalloprotease disclosed as SEQ ID NO: 2 in WO 2003/048353, or amino acids1-177 of SEQ ID NO: 2 in WO 2011/072191, with the following mutations:

D79L+S87P+A112P+D142L;

D79L+S87P+D142L; or

A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.

In another embodiment the protease is derived from a strain of thebacterium Pyrococcus, such as a strain of Pyrococcus furiosus (pfuprotease)

In an embodiment the protease is the one shown as SEQ ID NO: 1 in U.S.Pat. No. 6,358,726-B1. In another embodiment the protease is the oneshown as SEQ ID NO: 13 in WO2012/088303.

In another particular embodiment the composition comprises the hybridpullulanase of the invention, and a glucoamylase. In a specificembodiment the glucoamylase is from a strain of the genus Penicillium,especially a strain of Penicillium oxalicum, in particular thePenicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO2011/127802. In a preferred embodiment the glucoamylase is a variant ofthe Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO2011/127802 having a K79V substitution using the mature polypeptide(amino acids 22-616 of SEQ ID NO: 2) for numbering, and described in WO2013/036526. In a preferred embodiment the glucoamylase is a variant ofthe Penicillium oxalicum glucoamylase disclosed as amino acids 22-616 ofSEQ ID NO: 2 in WO 2011/127802, having a K79V substitution and one ormore of the following substitutions P2N, P4S, P11F, T65A, Q327F,especially P2N+P4S+P11F+T65A+Q327F as described in WO2013/053801.

In a specific embodiment the glucoamylase is from a strain of the genusPycnoporus, especially a strain of Pycnoporus sanguineus, in particularthe Pycnoporus sanguineus glucoamylase disclosed as SEQ ID NO: 2, 4, or6 in WO 2011/066576. In a preferred embodiment the enzyme compositioncomprises the glucoamylase shown as amino acids 19-573 of SEQ ID NO: 6in WO 2011/066576.

In a specific embodiment the glucoamylase is from a strain of the genusGloeophillum, especially a strain of Gloeophyllum trabeum, in particularthe Gloeophyllum trabeum glucoamylase disclosed as SEQ ID NO: 18 in WO2011/068803. In an especially preferred embodiment the enzymecomposition comprises the Gloeophyllum trabeum glucoamylase shown inamino acids 18-576 of SEQ ID NO: 18 in WO2011/068803, and having one ormore of the following substitutions: S95P, A121P, especially S95P+A121Pusing the mature polypeptide (positions 18-576 of SEQ ID NO: 18) fornumbering.

In a specific embodiment the glucoamylase is from a strain of the genusGloeophillum, especially a strain of Gloeophillum sepiarium, inparticular the mature Gloeophillum sepiarium glucoamylase disclosed asamino acids 18-573 of SEQ ID NO: 2 in WO2011/068803.

In a particular embodiment the composition comprises a variantpullulanase of the invention and a glucoamylase and optionally analpha-amylase, and wherein the pullulanase is selected from apolypeptide having at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the mature polypeptide of SEQ ID NO: 21 herein, and comprising thesubstitutions N368G+N393A+A492S, using SEQ ID NO: 3 for numbering, andthe glucoamylase is selected from i) a variant Gloeophyllum trabeumglucoamylase, which comprises the substitutions S95P+A121P compared tothe wild type Gloeophyllum trabeum glucoamylase amino acid sequence setforth in amino acids 18-576 of SEQ ID NO: 18 in WO 2011/068803; or ii) avariant having at least 85%, at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99% sequence identity to amino acids 18-576 of SEQID NO: 18 in WO 2011/068803, and the alpha-amylase is selected from: i)a variant Rhizomucor pusillus alpha-amylase with an Aspergillus nigerglucoamylase linker and starch-binding domain (SBD), which comprises thesubstitutions G128D+D143N compared to the hybrid Rhizomucor pusillusalpha-amylase amino acid sequence set forth in SEQ ID NO: 7 inWO2013/006756; or ii) a variant having at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% sequence identity tothe polypeptide of SEQ ID NO: 7 in WO2013/006756.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Forinstance, the composition may be in the form of granulate ormicrogranulate. The variant may be stabilized in accordance with methodsknown in the art.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

The enzyme composition of the present invention may be in any formsuitable for use, such as, for example, a crude fermentation broth withor without cells removed, a cell lysate with or without cellular debris,a semi-purified or purified enzyme composition, or a host cell, e.g.,Trichoderma host cell, as a source of the enzymes.

The enzyme composition may be a dry powder or granulate, a non-dustinggranulate, a liquid, a stabilized liquid, or a stabilized protectedenzyme. Liquid enzyme compositions may, for instance, be stabilized byadding stabilizers such as a sugar, a sugar alcohol or another polyol,and/or lactic acid or another organic acid according to establishedprocesses.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Methods of Using the Pullulanase Variants—Industrial Applications

The present invention is also directed to methods of using polypeptideof present invention in various industrial applications.

The polypeptide of the present invention may be used for starchprocesses, in particular starch conversion, especially liquefaction ofstarch (see, e.g., U.S. Pat. No. 3,912,590, EP 252730 and EP 063909, WO99/19467, and WO 96/28567, which are all hereby incorporated byreference). Also contemplated are compositions for starch conversionpurposes, which may beside the polypeptide of the present invention alsocomprise a glucoamylase (AMG), and an alpha-amylase.

Further, the polypeptide of the present invention is particularly usefulin the production of sweeteners and ethanol (see, e.g., U.S. Pat. No.5,231,017, which is hereby incorporated by reference), such as fuel,drinking and industrial ethanol, from starch or whole grains. In oneembodiment the present invention relates to a use of the polypeptideaccording to the invention for production of a syrup and/or afermentation product from a starch containing material. The starchmaterial may in one embodiment be gelatinized. In another embodiment thestarch material is ungelatinized.

Starch Processing

Native starch consists of microscopic granules, which are insoluble inwater at room temperature. When an aqueous starch slurry is heated, thegranules swell and eventually burst, dispersing the starch moleculesinto the solution. At temperatures up to about 50° C. to 75° C. theswelling may be reversible. However, with higher temperatures anirreversible swelling called “gelatinization” begins. During this“gelatinization” process there is a dramatic increase in viscosity.Granular starch to be processed may be a highly refined starch quality,preferably at least 90%, at least 95%, at least 97% or at least 99.5%pure or it may be a more crude starch-containing materials comprising(e.g., milled) whole grains including non-starch fractions such as germresidues and fibers. The raw material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure and allowing for further processing. In dry milling wholekernels are milled and used. Wet milling gives a good separation of germand meal (starch granules and protein) and is often applied at locationswhere the starch hydrolyzate is used in the production of, e.g., syrups.Both dry and wet milling is well known in the art of starch processingand may be used in a process of the invention. Methods for reducing theparticle size of the starch containing material are well known to thoseskilled in the art.

As the solids level is 30-40% in a typical industrial process, thestarch has to be thinned or “liquefied” so that it can be suitablyprocessed. This reduction in viscosity is primarily attained byenzymatic degradation in current commercial practice.

Liquefaction is carried out in the presence of an alpha-amylase,preferably a bacterial alpha-amylase and/or acid fungal alpha-amylase.In an embodiment, a phytase is also present during liquefaction. In anembodiment, viscosity reducing enzymes such as a xylanase and/orbeta-glucanase is also present during liquefaction.

During liquefaction, the long-chained starch is degraded into branchedand linear shorter units (maltodextrins) by an alpha-amylase.Liquefaction may be carried out as a three-step hot slurry process. Theslurry is heated to between 60-95° C. (e.g., 70-90° C., such as 77-86°C., 80-85° C., 83-85° C.) and an alpha-amylase is added to initiateliquefaction (thinning).

The slurry may in an embodiment be jet-cooked at between 95-140° C.,e.g., 105-125° C., for about 1-15 minutes, e.g., about 3-10 minutes,especially around 5 minutes. The slurry is then cooled to 60-95° C. andmore alpha-amylase is added to obtain final hydrolysis (secondaryliquefaction). The jet-cooking process is carried out at pH 4.5-6.5,typically at a pH between 5 and 6. The alpha-amylase may be added as asingle dose, e.g., before jet cooking.

The liquefaction process is carried out at between 70-95° C., such as80-90° C., such as around 85° C., for about 10 minutes to 5 hours,typically for 1-2 hours. The pH is between 4 and 7, such as between 5.5and 6.2. In order to ensure optimal enzyme stability under theseconditions, calcium may optionally be added (to provide 1-60 ppm freecalcium ions, such as about 40 ppm free calcium ions). After suchtreatment, the liquefied starch will typically have a “dextroseequivalent” (DE) of 10-15.

Generally liquefaction and liquefaction conditions are well known in theart. Examples of alpha-amylase are disclosed in the “Alpha-Amylases”section below. Saccharification may be carried out using conditionswell-known in the art with a carbohydrate-source generating enzyme, inparticular a glucoamylase, or a beta-amylase and optionally adebranching enzyme, such as an isoamylase or a pullulanase. Forinstance, a full saccharification step may last from about 24 to about72 hours. However, it is common to do a pre-saccharification oftypically 40-90 minutes at a temperature between 30-65° C., typicallyabout 60° C., followed by complete saccharification during fermentationin a simultaneous saccharification and fermentation (SSF) process.Saccharification is typically carried out at a temperature in the rangeof 20-75° C., e.g., 25-65° C. and 40-70° C., typically around 60° C.,and at a pH between about 4 and 5, normally at about pH 4.5.

The saccharification and fermentation steps may be carried out eithersequentially or simultaneously. In an embodiment, saccharification andfermentation are performed simultaneously (referred to as “SSF”).However, it is common to perform a pre-saccharification step for about30 minutes to 2 hours (e.g., 30 to 90 minutes) at a temperature of 30 to65° C., typically around 60° C. which is followed by a completesaccharification during fermentation referred to as simultaneoussaccharification and fermentation (SSF). The pH is usually between4.2-4.8, e.g., pH 4.5. In a simultaneous saccharification andfermentation (SSF) process, there is no holding stage forsaccharification, rather, the yeast and enzymes are added together.

In a typical saccharification process, maltodextrins produced duringliquefaction are converted into dextrose by adding a glucoamylase and adebranching enzyme, such as an isoamylase (U.S. Pat. No. 4,335,208) or apullulanase. The temperature is lowered to 60° C., prior to the additionof the glucoamylase and debranching enzyme. The saccharification processproceeds for 24-72 hours. Prior to addition of the saccharifyingenzymes, the pH is reduced to below 4.5, while maintaining a hightemperature (above 95° C.), to inactivate the liquefying alpha-amylase.This process reduces the formation of short oligosaccharide called“panose precursors,” which cannot be hydrolyzed properly by thedebranching enzyme. Normally, about 0.2-0.5% of the saccharificationproduct is the branched trisaccharide panose (GIc pα1-6Glc pα1-4Glc),which cannot be degraded by a pullulanase. If active amylase from theliquefaction remains present during saccharification (i.e., nodenaturing), the amount of panose can be as high as 1-2%, which ishighly undesirable since it lowers the saccharification yieldsignificantly.

Other fermentation products may be fermented at conditions andtemperatures well known to persons skilled in the art, suitable for thefermenting organism in question. The fermentation product may berecovered by methods well known in the art, e.g., by distillation.Examples of carbohydrate-source generating enzymes are disclosed in the“Enzymes” section below.

In a particular embodiment, the process of the invention furthercomprises, prior to the conversion of a starch-containing material tosugars/dextrins the steps of:

(x) reducing the particle size of the starch-containing material; and

(y) forming a slurry comprising the starch-containing material andwater.

In an embodiment, the starch-containing material is milled to reduce theparticle size. In an embodiment the particle size is reduced to between0.05-3.0 mm, preferably 0.1-0.5 mm, or so that at least 30%, preferablyat least 50%, more preferably at least 70%, even more preferably atleast 90% of the starch-containing material fits through a sieve with a0.05-3.0 mm screen, preferably 0.1-0.5 mm screen.

The aqueous slurry may contain from 10-55 wt. % dry solids (DS),preferably 25-45 wt. % dry solids (DS), more preferably 30-40 wt. % drysolids (DS) of starch-containing material. Conventionalstarch-conversion processes, such as liquefaction and saccharificationprocesses are described, e.g., in U.S. Pat. No. 3,912,590, EP 252730 andEP 063909, which are incorporated herein by reference.

In an embodiment, the conversion process degrading starch to lowermolecular weight carbohydrate components such as sugars or fat replacersincludes a debranching step. In the case of converting starch into asugar, the starch is depolymerized. Such a depolymerization processconsists of, e.g., a pre-treatment step and two or three consecutiveprocess steps, i.e., a liquefaction process, a saccharification process,and depending on the desired end-product, an optional isomerizationprocess.

When the desired final sugar product is, e.g., high fructose syrup thedextrose syrup may be converted into fructose. After thesaccharification process, the pH is increased to a value in the range of6-8, e.g., pH 7.5, and the calcium is removed by ion exchange. Thedextrose syrup is then converted into high fructose syrup using, e.g.,an immobilized glucose isomerase.

Production of Fermentation Products

Fermentable sugars (e.g., dextrins, monosaccharides, particularlyglucose) are produced from enzymatic saccharification. These fermentablesugars may be further purified and/or converted to useful sugarproducts. In addition, the sugars may be used as a fermentationfeedstock in a microbial fermentation process for producingend-products, such as alcohol (e.g., ethanol, and butanol), organicacids (e.g., succinic acid, 3-HP and lactic acid), sugar alcohols (e.g.,glycerol), ascorbic acid intermediates (e.g., gluconate,2-keto-D-gluconate, 2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid),amino acids (e.g., lysine), proteins (e.g., antibodies and fragmentthereof).

In an embodiment, the fermentable sugars obtained during theliquefaction process steps are used to produce alcohol and particularlyethanol. In ethanol production, an SSF process is commonly used whereinthe saccharifying enzymes and fermenting organisms (e.g., yeast) areadded together and then carried out at a temperature of 30-40° C.

The organism used in fermentation will depend on the desiredend-product. Typically, if ethanol is the desired end product yeast willbe used as the fermenting organism. In some preferred embodiments, theethanol-producing microorganism is a yeast and specificallySaccharomyces such as strains of S. cerevisiae (U.S. Pat. No.4,316,956). A variety of S. cerevisiae are commercially available andthese include but are not limited to FALl (Fleischmann's Yeast),SUPERSTART (Alltech), FERMIOL (DSM Specialties), RED STAR (Lesaffre) andAngel alcohol yeast (Angel Yeast Company, China). The amount of starteryeast employed in the methods is an amount effective to produce acommercially significant amount of ethanol in a suitable amount of time,(e.g., to produce at least 10% ethanol from a substrate having between25-40% DS in less than 72 hours). Yeast cells are generally supplied inamounts of about 10⁴ to about 10¹², and preferably from about 10⁷ toabout 10¹⁰ viable yeast count per mL of fermentation broth. After yeastis added to the mash, it is typically subjected to fermentation forabout 24-96 hours, e.g., 35-60 hours. The temperature is between about26-34° C., typically at about 32° C., and the pH is from pH 3-6, e.g.,around pH 4-5.

The fermentation may include, in addition to a fermenting microorganisms(e.g., yeast), nutrients, and additional enzymes, including phytases.The use of yeast in fermentation is well known in the art.

In further embodiments, use of appropriate fermenting microorganisms, asis known in the art, can result in fermentation end product including,e.g., glycerol, 1,3-propanediol, gluconate, 2-keto-D-gluconate,2,5-diketo-D-gluconate, 2-keto-L-gulonic acid, succinic acid, lacticacid, amino acids, and derivatives thereof. More specifically whenlactic acid is the desired end product, a Lactobacillus sp. (L. casei)may be used; when glycerol or 1,3-propanediol are the desiredend-products E. coli may be used; and when 2-keto-D-gluconate,2,5-diketo-D-gluconate, and 2-keto-L-gulonic acid are the desired endproducts, Pantoea citrea may be used as the fermenting microorganism.The above enumerated list are only examples and one skilled in the artwill be aware of a number of fermenting microorganisms that may be usedto obtain a desired end product.

Processes for Producing Fermentation Products from UngelatinizedStarch-Containing Material

The invention relates to processes for producing fermentation productsfrom starch-containing material without gelatinization (i.e., withoutcooking) of the starch-containing material (often referred to as a “rawstarch hydrolysis” process). The fermentation product, such as ethanol,can be produced without liquefying the aqueous slurry containing thestarch-containing material and water. In one embodiment a process of theinvention includes saccharifying (e.g., milled) starch-containingmaterial, e.g., granular starch, below the initial gelatinizationtemperature, preferably in the presence of alpha-amylase and/orcarbohydrate-source generating enzyme(s) to produce sugars that can befermented into the fermentation product by a suitable fermentingorganism. In this embodiment the desired fermentation product, e.g.,ethanol, is produced from ungelatinized (i.e., uncooked), preferablymilled, cereal grains, such as corn.

Accordingly, in one aspect the invention relates to processes forproducing fermentation products from starch-containing materialcomprising simultaneously saccharifying and fermenting starch-containingmaterial using a carbohydrate-source generating enzyme and a fermentingorganism at a temperature below the initial gelatinization temperatureof said starch-containing material. Saccharification and fermentationmay also be separate. Thus in another aspect the invention relates toprocesses of producing fermentation products, comprising the followingsteps:

(i) saccharifying a starch-containing material at a temperature belowthe initial gelatinization temperature; and(ii) fermenting using a fermentation organism;wherein step (i) is carried out using at least a glucoamylase, and avariant pullulanase according to the invention.

In one embodiment, an alpha amylase is added in step (i). In anotherembodiment steps (i) and

(ii) are performed simultaneously.

In one embodiment, a protease is also present. The protease may be anyacid fungal protease or metalloprotease. The fermentation product, e.g.,ethanol, may optionally be recovered after fermentation, e.g., bydistillation. Typically amylase(s), such as glucoamylase(s) and/or othercarbohydrate-source generating enzymes, and/or alpha-amylase(s), is(are)present during fermentation. Examples of glucoamylases and othercarbohydrate-source generating enzymes include raw starch hydrolyzingglucoamylases. Examples of alpha-amylase(s) include acid alpha-amylasessuch as acid fungal alpha-amylases. Examples of fermenting organismsinclude yeast, e.g., a strain of Saccharomyces cerevisiae. The term“initial gelatinization temperature” means the lowest temperature atwhich starch gelatinization commences. In general, starch heated inwater begins to gelatinize between about 50° C. and 75° C.; the exacttemperature of gelatinization depends on the specific starch and canreadily be determined by the skilled artisan. Thus, the initialgelatinization temperature may vary according to the plant species, tothe particular variety of the plant species as well as with the growthconditions. In the context of this invention the initial gelatinizationtemperature of a given starch-containing material may be determined asthe temperature at which birefringence is lost in 5% of the starchgranules using the method described by Gorinstein and Lii, 1992,Starch/Stärke 44(12): 461-466. Before initiating the process a slurry ofstarch-containing material, such as granular starch, having 10-55 w/w %dry solids (DS), preferably 25-45 w/w % dry solids, more preferably30-40 w/w % dry solids of starch-containing material may be prepared.The slurry may include water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants. Because the process of the invention iscarried out below the initial gelatinization temperature, and thus nosignificant viscosity increase takes place, high levels of stillage maybe used if desired. In an embodiment the aqueous slurry contains fromabout 1 to about 70 vol. %, preferably 15-60 vol. %, especially fromabout 30 to 50 vol. % water and/or process waters, such as stillage(backset), scrubber water, evaporator condensate or distillate,side-stripper water from distillation, or process water from otherfermentation product plants, or combinations thereof, or the like. Thestarch-containing material may be prepared by reducing the particlesize, preferably by dry or wet milling, to 0.05 to 3.0 mm, preferably0.1-0.5 mm. After being subjected to a process of the invention at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or preferably at least99% of the dry solids in the starch-containing material are convertedinto a soluble starch hydrolyzate. A process in this aspect of theinvention is conducted at a temperature below the initial gelatinizationtemperature, which means that the temperature typically lies in therange between 30-75° C., preferably between 45-60° C. In a preferredembodiment the process carried at a temperature from 25° C. to 40° C.,such as from 28° C. to 35° C., such as from 30° C. to 34° C., preferablyaround 32° C. In an embodiment the process is carried out so that thesugar level, such as glucose level, is kept at a low level, such asbelow 6 w/w %, such as below about 3 w/w %, such as below about 2 w/w %,such as below about 1 w/w %, such as below about 0.5 w/w %, or below0.25 w/w %, such as below about 0.1 w/w %. Such low levels of sugar canbe accomplished by simply employing adjusted quantities of enzyme andfermenting organism. A skilled person in the art can easily determinewhich doses/quantities of enzyme and fermenting organism to use. Theemployed quantities of enzyme and fermenting organism may also beselected to maintain low concentrations of maltose in the fermentationbroth. For instance, the maltose level may be kept below about 0.5 w/w%, such as below about 0.2 w/w %. The process of the invention may becarried out at a pH from about 3 and 7, preferably from pH 3.5 to 6, ormore preferably from pH 4 to 5. In an embodiment fermentation is ongoingfor 6 to 120 hours, in particular 24 to 96 hours.

Processes for Producing Fermentation Products from GelatinizedStarch-Containing Material

In this aspect, the invention relates to processes for producingfermentation products, especially ethanol, from starch-containingmaterial, which process includes a liquefaction step and sequentially orsimultaneously performed saccharification and fermentation steps.Consequently, the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

(a) liquefying starch-containing material in the presence of analpha-amylase; or(b) saccharifying the liquefied material obtained in step (a) using aglucoamylase;(c) fermenting using a fermenting organism;wherein step (a) and/or step (b) is carried out in the presence of apullulanase according to the invention.

In an embodiment, a protease, such as an acid fungal protease or ametallo protease is added before, during and/or after liquefaction. Inan embodiment the metalloprotease is derived from a strain ofThermoascus, e.g., a strain of Thermoascus aurantiacus, especiallyThermoascus aurantiacus CGMCC No. 0670. In another embodiment theprotease is a bacterial protease, particularly a protease derived from astrain of Pyrococcus, more particularly from Pyrococcus furiosusdisclosed in U.S. Pat. No. 6,358,726. In an embodiment the glucoamylasederived from a strain of Aspergillus, e.g., Aspergillus niger orAspergillus awamori, a strain of Talaromyces, especially Talaromycesemersonii; or a strain of Athelia, especially Athelia rolfsii; a strainof Trametes, e.g., Trametes cingulata; a strain of the genusGloeophyllum, e.g., a strain of Gloeophyllum sepiarum or Gloeophyllumtrabeum; or a mixture thereof. Saccharification step (b) andfermentation step (c) may be carried out either sequentially orsimultaneously. A pullulanase and/or metalloprotease may be added duringsaccharification and/or fermentation when the process is carried out asa sequential saccharification and fermentation process and before orduring fermentation when steps (b) and (c) are carried outsimultaneously (SSF process). The pullulanase and/or metalloprotease mayalso advantageously be added before liquefaction (pre-liquefactiontreatment), i.e., before or during step (a), and/or after liquefaction(post liquefaction treatment), i.e., after step (a). The pullulanase ismost advantageously added before or during liquefaction, i.e., before orduring step (a). The fermentation product, such as especially ethanol,may optionally be recovered after fermentation, e.g., by distillation.The fermenting organism is preferably yeast, preferably a strain ofSaccharomyces cerevisiae. In a particular embodiment, the process of theinvention further comprises, prior to step (a), the steps of:

x) reducing the particle size of the starch-containing material,preferably by milling (e.g., using a hammer mill);y) forming a slurry comprising the starch-containing material and water.

In an embodiment, the particle size is smaller than a #7 screen, e.g., a#6 screen. A #7 screen is usually used in conventional prior artprocesses. The aqueous slurry may contain from 10-55, e.g., 25-45 and30-40, w/w % dry solids (DS) of starch-containing material. The slurryis heated to above the gelatinization temperature and an alpha-amylasevariant may be added to initiate liquefaction (thinning). The slurry mayin an embodiment be jet-cooked to further gelatinize the slurry beforebeing subjected to alpha-amylase in step (a). Liquefaction may in anembodiment be carried out as a three-step hot slurry process. The slurryis heated to between 60-95° C., preferably between 70-90° C., such aspreferably between 80-85° C. at pH 4-6, preferably 4.5-5.5, andalpha-amylase variant, optionally together with a pullulanase and/orprotease, preferably metalloprotease, are added to initiate liquefaction(thinning). In an embodiment the slurry may then be jet-cooked at atemperature between 95-140° C., preferably 100-135° C., such as 105-125°C., for about 1-15 minutes, preferably for about 3-10 minutes,especially around about 5 minutes. The slurry is cooled to 60-95° C. andmore alpha-amylase variant and optionally pullulanase variant and/orprotease, preferably metalloprotease, is(are) added to finalizehydrolysis (secondary liquefaction). The liquefaction process is usuallycarried out at pH 4.0-6, in particular at a pH from 4.5 to 5.5.Saccharification step (b) may be carried out using conditions well knownin the art. For instance, a full saccharification process may last up tofrom about 24 to about 72 hours, however, it is common only to do apre-saccharification of typically 40-90 minutes at a temperature between30-65° C., typically about 60° C., followed by complete saccharificationduring fermentation in a simultaneous saccharification and fermentationprocess (SSF process). Saccharification is typically carried out attemperatures from 20-75° C., preferably from 40-70° C., typically around60° C., and at a pH between 4 and 5, normally at about pH 4.5. The mostwidely used process to produce a fermentation product, especiallyethanol, is a simultaneous saccharification and fermentation (SSF)process, in which there is no holding stage for the saccharification,meaning that a fermenting organism, such as yeast, and enzyme(s), may beadded together. SSF may typically be carried out at a temperature from25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to34° C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours.

Starch-Containing Materials

Any suitable starch-containing starting material may be used in aprocess of the present invention. The starting material is generallyselected based on the desired fermentation product. Examples ofstarch-containing starting materials, suitable for use in the processesof the present invention, include barley, beans, cassava, cereals, corn,milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca,wheat, and whole grains, or any mixture thereof. The starch-containingmaterial may also be a waxy or non-waxy type of corn and barley. In apreferred embodiment the starch-containing material is corn. In apreferred embodiment the starch-containing material is wheat.

Fermentation Products

The term “fermentation product” means a product produced by a method orprocess including fermenting using a fermenting organism. Fermentationproducts include alcohols (e.g., ethanol, methanol, butanol); organicacids (e.g., citric acid, acetic acid, itaconic acid, lactic acid,succinic acid, gluconic acid); ketones (e.g., acetone); amino acids(e.g., glutamic acid); gases (e.g., H₂ and CO₂); antibiotics (e.g.,penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B₁₂,beta-carotene); and hormones. In a preferred embodiment the fermentationproduct is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potableneutral spirits; or industrial ethanol or products used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry and tobacco industry.Preferred beer types comprise ales, stouts, porters, lagers, bitters,malt liquors, happoushu, high-alcohol beer, low-alcohol beer,low-calorie beer or light beer. In an preferred embodiment thefermentation product is ethanol.

Starch Slurry Processing with Stillage

Milled starch-containing material is combined with water and recycledthin-stillage resulting in an aqueous slurry. The slurry can comprisebetween 15 to 55% ds w/w (e.g., 20 to 50%, 25 to 50%, 25 to 45%, 25 to40%, 20 to 35% and 30-36% ds). In some embodiments, the recycledthin-stillage (backset) is in the range of about 10 to 70% v/v (e.g., 10to 60%, 10 to 50%, 10 to 40%, 10 to 30%, 10 to 20%, 20 to 60%, 20 to50%, 20 to 40% and also 20 to 30%).

Once the milled starch-containing material is combined with water andbackset, the pH is not adjusted in the slurry. Further the pH is notadjusted after the addition of a phytase and optionally an alpha-amylaseto the slurry. In an embodiment, the pH of the slurry will be in therange of about pH 4.5 to less than about 6.0 (e.g., pH 4.5 to 5.8, pH4.5 to 5.6, pH 4.8 to 5.8, pH 5.0 to 5.8, pH 5.0 to 5.4, pH 5.2 to 5.5and pH 5.2 to 5.9). The pH of the slurry may be between about pH 4.5 and5.2 depending on the amount of thin stillage added to the slurry and thetype of material comprising the thin stillage. For example, the pH ofthe thin stillage may be between pH 3.8 and pH 4.5.

During ethanol production, acids can be added to lower the pH in thebeer well, to reduce the risk of microbial contamination prior todistillation.

In some embodiments, a phytase is added to the slurry. In otherembodiments, in addition to phytase, an alpha-amylase is added to theslurry. In some embodiments, a phytase and alpha-amylase are added tothe slurry sequentially. In other embodiments, a phytase andalpha-amylase are added simultaneously. In some embodiments, the slurrycomprising a phytase and optionally, an alpha-amylase, are incubated(pretreated) for a period of about 5 minutes to about 8 hours (e.g., 5minutes to 6 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, and 15minutes to 4 hours). In other embodiments, the slurry is incubated at atemperature in the range of about 40 to 115° C. (e.g., 45 to 80° C., 50to 70° C., 50 to 75° C., 60 to 110° C., 60 to 95° C., 70 to 110° C., 70to 85° C. and 77 to 86° C.).

In other embodiments, the slurry is incubated at a temperature of about0 to about 30° C. (e.g., 0 to 25° C., 0 to 20° C., 0 to 15° C., 0 to 10°C. and 0 to 5° C.) below the starch gelatinization temperature of thestarch-containing material. In some embodiments, the temperature isbelow about 68° C., below about 65° C., below about 62° C., below about60° C. and below about 55° C. In some embodiments, the temperature isabove about 45° C., above about 50° C., above about 55° C. and aboveabout 60° C. In some embodiments, the incubation of the slurrycomprising a phytase and an alpha-amylase at a temperature below thestarch gelatinization temperature is referred to as a primary (1°)liquefaction.

In one embodiment, the milled starch-containing material is corn ormilo. The slurry comprises 25 to 40% DS, the pH is in the range of 4.8to 5.2, and the slurry is incubated with a phytase and optionally analpha-amylase for 5 minutes to 2 hours, at a temperature range of 60 to75° C.

Currently, it is believed that commercially-available microbialalpha-amylases used in the liquefaction process are generally not stableenough to produce liquefied starch substrate from a dry mill processusing whole ground grain at a temperature above about 80° C. at a pHlevel that is less than pH 5.6. The stability of many commerciallyavailable alpha-amylases is reduced at a pH of less than about 4.0.

In a further liquefaction step, the incubated or pretreatedstarch-containing material is exposed to an increase in temperature suchas about 0 to about 45° C. above the starch gelatinization temperatureof the starch-containing material (e.g., 70° C. to 120° C., 70° C. to110° C., and 70° C. to 90° C.) for a period of time of about 2 minutesto about 6 hours (e.g., 2 minutes to 4 hours, 90 minutes, 140 minutesand 90 to 140 minutes) at a pH of about 4.0 to 5.5 more preferablybetween 1 hour to 2 hours. The temperature can be increased by aconventional high temperature jet cooking system for a short period oftime, for example, for 1 to 15 minutes. Then the starch may be furtherhydrolyzed at a temperature ranging from about 75° C. to 95° C. (e.g.,80° C. to 90° C. and 80° C. to 85° C.) for a period of about 15 to 150minutes (e.g., 30 to 120 minutes). In a preferred embodiment, the pH isnot adjusted during these process steps and the pH of the liquefied mashis in the range of about pH 4.0 to pH 5.8 (e.g., pH 4.5 to 5.8, pH 4.8to 5.4, and pH 5.0 to 5.2). In some embodiments, a second dose ofthermostable alpha-amylase is added to the secondary liquefaction step,but in other embodiments there is no additional dosage of alpha-amylase.

The incubation and liquefaction steps may be followed bysaccharification and fermentation steps well known in the art.

Distillation

Optionally, following fermentation, an alcohol (e.g., ethanol) may beextracted by, for example, distillation and optionally followed by oneor more process steps.

In some embodiments, the yield of ethanol produced by the methodsprovided herein is at least 8%, at least 10%, at least 12%, at least14%, at least 15%, at least 16%, at least 17% and at least 18% (v/v) andat least 23% v/v. The ethanol obtained according to the process providedherein may be used as, for example, fuel ethanol, drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

By-Products

Left over from the fermentation is the grain, which is typically usedfor animal feed either in liquid or dried form. In further embodiments,the end product may include the fermentation co-products such asdistiller's dried grains (DDG) and distiller's dried grain plus solubles(DDGS), which may be used, for example, as an animal feed.

Further details on how to carry out liquefaction, saccharification,fermentation, distillation, and recovery of ethanol are well known tothe skilled person.

According to the process provided herein, the saccharification andfermentation may be carried out simultaneously or separately.

Fermenting Organisms

The term “fermenting organism” refers to any organism, includingbacterial and fungal organisms, such as yeast and filamentous fungi,suitable for producing a desired fermentation product. Suitablefermenting organisms are able to ferment, i.e., convert, fermentablesugars, such as arabinose, fructose, glucose, maltose, mannose, orxylose, directly or indirectly into the desired fermentation product.

Examples of fermenting organisms include fungal organisms such as yeast.Preferred yeast include strains of Saccharomyces, in particularSaccharomyces cerevisiae or Saccharomyces uvarum; strains of Pichia, inparticular Pichia stipitis such as Pichia stipitis CBS 5773 or Pichiapastoris; strains of Candida, in particular Candida arabinofermentans,Candida boidinii, Candida diddensii, Candida shehatae, Candidasonorensis, Candida tropicalis, or Candida utilis. Other fermentingorganisms include strains of Hansenula, in particular Hansenula anomalaor Hansenula polymorpha; strains of Kluyveromyces, in particularKluyveromyces fragilis or Kluyveromyces marxianus; and strains ofSchizosaccharomyces, in particular Schizosaccharomyces pombe.

Preferred bacterial fermenting organisms include strains of Escherichia,in particular Escherichia coli, strains of Zymomonas, in particularZymomonas mobilis, strains of Zymobacter, in particular Zymobactorpalmae, strains of Klebsiella in particular Klebsiella oxytoca, strainsof Leuconostoc, in particular Leuconostoc mesenteroides, strains ofClostridium, in particular Clostridium butyricum, strains ofEnterobacter, in particular Enterobacter aerogenes, and strains ofThermoanaerobacter, in particular Thermoanaerobacter BG1L1 (Appl.Microbiol. Biotech. 77: 61-86), Thermoanarobacter ethanolicus,Thermoanaerobacter mathranii, or Thermoanaerobacterthermosaccharolyticum. Strains of Lactobacillus are also envisioned asare strains of Corynebacterium glutamicum R, Bacillusthermoglucosidaisus, and Geobacillus thermoglucosidasius.

In an embodiment, the fermenting organism is a C6 sugar fermentingorganism, such as a strain of, e.g., Saccharomyces cerevisiae.

In an embodiment, the fermenting organism is a C5 sugar fermentingorganism, such as a strain of, e.g., Saccharomyces cerevisiae.

In one embodiment, the fermenting organism is added to the fermentationmedium so that the viable fermenting organism, such as yeast, count permL of fermentation medium is in the range from 10⁵ to 10¹², preferablyfrom 10⁷ to 10¹⁰, especially about 5×10⁷.

Yeast is the preferred fermenting organism for ethanol fermentation.Preferred are strains of Saccharomyces, especially strains of thespecies Saccharomyces cerevisiae, preferably strains which are resistanttowards high levels of ethanol, i.e., up to, e.g., about 10, 12, 15 or20 vol. % or more ethanol.

In an embodiment, the C5 utilizing yeast is a Saccharomyces cereviseastrain disclosed in WO 2004/085627.

In an embodiment, the fermenting organism is a C5 eukaryotic microbialcell concerned in WO 2010/074577 (Nedalco).

In an embodiment, the fermenting organism is a transformed C5 eukaryoticcell capable of directly isomerize xylose to xylose disclosed in US2008/0014620.

In an embodiment, the fermenting organism is a C5 sugar fermentatingcell disclosed in WO 2009/109633.

Commercially available yeast include LNF SA-1, LNF BG-1, LNF PE-2, andLNF CAT-1 (available from LNF Brazil), RED STAR™ and ETHANOL RED™ yeast(available from Fermentis/Lesaffre, USA), FALl (available fromFleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast(available from Ethanol Technology, WI, USA), BIOFERM AFT and XR(available from NABC—North American Bioproducts Corporation, GA, USA),GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

The fermenting organism capable of producing a desired fermentationproduct from fermentable sugars is preferably grown under preciseconditions at a particular growth rate. When the fermenting organism isintroduced into/added to the fermentation medium the inoculatedfermenting organism pass through a number of stages. Initially growthdoes not occur. This period is referred to as the “lag phase” and may beconsidered a period of adaptation. During the next phase referred to asthe “exponential phase” the growth rate gradually increases. After aperiod of maximum growth the rate ceases and the fermenting organismenters “stationary phase”. After a further period of time the fermentingorganism enters the “death phase” where the number of viable cellsdeclines.

Fermentation

The fermentation conditions are determined based on, e.g., the kind ofplant material, the available fermentable sugars, the fermentingorganism(s) and/or the desired fermentation product. One skilled in theart can easily determine suitable fermentation conditions. Thefermentation may be carried out at conventionally used conditions.Preferred fermentation processes are anaerobic processes.

For example, fermentations may be carried out at temperatures as high as75° C., e.g., between 40-70° C., such as between 50-60° C. However,bacteria with a significantly lower temperature optimum down to aroundroom temperature (around 20° C.) are also known. Examples of suitablefermenting organisms can be found in the “Fermenting Organisms” sectionabove.

For ethanol production using yeast, the fermentation may go on for 24 to96 hours, in particular for 35 to 60 hours. In an embodiment thefermentation is carried out at a temperature between 20 to 40° C.,preferably 26 to 34° C., in particular around 32° C. In an embodimentthe pH is from pH 3 to 6, preferably around pH 4 to 5.

Other fermentation products may be fermented at temperatures known tothe skilled person in the art to be suitable for the fermenting organismin question.

Fermentation is typically carried out at a pH in the range between 3 and7, preferably from pH 3.5 to 6, such as around pH 5. Fermentations aretypically ongoing for 6-96 hours.

The processes of the invention may be performed as a batch or as acontinuous process. Fermentations may be conducted in an ultrafiltrationsystem wherein the retentate is held under recirculation in the presenceof solids, water, and the fermenting organism, and wherein the permeateis the desired fermentation product containing liquid. Equallycontemplated are methods/processes conducted in continuous membranereactors with ultrafiltration membranes and where the retentate is heldunder recirculation in presence of solids, water, and the fermentingorganism(s) and where the permeate is the fermentation productcontaining liquid.

After fermentation the fermenting organism may be separated from thefermented slurry and recycled.

Fermentation Medium

The phrase “fermentation media” or “fermentation medium” refers to theenvironment in which fermentation is carried out and comprises thefermentation substrate, that is, the carbohydrate source that ismetabolized by the fermenting organism(s).

The fermentation medium may comprise other nutrients and growthstimulator(s) for the fermenting organism(s). Nutrient and growthstimulators are widely used in the art of fermentation and includenitrogen sources, such as ammonia; vitamins and minerals, orcombinations thereof.

Recovery

Subsequent to fermentation, the fermentation product may be separatedfrom the fermentation medium. The fermentation medium may be distilledto extract the desired fermentation product or the desired fermentationproduct may be extracted from the fermentation medium by micro ormembrane filtration techniques. Alternatively, the fermentation productmay be recovered by stripping. Methods for recovery are well known inthe art.

Compositions

The present invention also relates to compositions comprising a hybridpullulanase according to the invention and at least one additionalenzyme. The additional enzyme(s) may be selected from the groupconsisting of an alpha amylase, glucoamylase, beta-amylase, cellulase(beta-glucosidase, cellobiohydrolase and endoglucanase), hemicellulase(e.g., xylanase), isoamylase, isomerase, lipase, phytase, protease, afurther pullulanase, and/or other enzymes useful in a commercial processin conjunction with a pullulanase. Such enzymes are known in the art instarch processing, sugar conversion, fermentations for alcohol and otheruseful end-products, commercial detergents and cleaning aids, stainremoval, fabric treatment or desizing, and the like.

The enzyme composition of the present invention may be in any formsuitable for use, such as, for example, a crude fermentation broth withor without cells removed, a cell lysate with or without cellular debris,a semi-purified or purified enzyme composition, or a host cell, e.g.,Trichoderma host cell, as a source of the enzymes.

The enzyme composition may be a dry powder or granulate, a non-dustinggranulate, a liquid, a stabilized liquid, or a stabilized protectedenzyme. Liquid enzyme compositions may, for instance, be stabilized byadding stabilizers such as a sugar, a sugar alcohol or another polyol,and/or lactic acid or another organic acid according to establishedprocesses.

Enzymes

The enzyme(s) and polypeptides described below are to be used in an“effective amount” in processes of the present invention.

Alpha-Amylases

Any alpha-amylase may be used, such as of fungal, bacterial or plantorigin. In a preferred embodiment the alpha-amylase is an acidalpha-amylase, e.g., acid fungal or acid bacterial alpha-amylase. Theterm “acid alpha-amylase” means an alpha-amylase (EC 3.2.1.1) whichadded in an effective amount has activity optimum at a pH in the rangeof 3 to 7, preferably from 3.5 to 6, or more preferably from 4-5.

Bacterial Alpha-Amylases

An alpha-amylase for use in the present invention may be a bacterialalpha-amylase, e.g., derived from Bacillus. In a preferred embodimentthe Bacillus alpha-amylase is derived from a strain of Bacillusamyloliquefaciens, Bacillus licheniformis, Bacillus stearothermophilus,or Bacillus subtilis, but may also be derived from other Bacillus sp.

Specific examples of alpha-amylases include the Bacillusamyloliquefaciens alpha-amylase of SEQ ID NO: 5 in WO 99/19467, theBacillus licheniformis alpha-amylase of SEQ ID NO: 4 in WO 99/19467, andthe Bacillus stearothermophilus alpha-amylase of SEQ ID NO: 3 in WO99/19467 (all sequences are hereby incorporated by reference). In anembodiment the alpha-amylase may be an enzyme having a degree ofidentity of at least 60%, e.g., at least 70%, at least 80%, at least90%, at least 95%, at least 96%, at least 97%, at least 98% or at least99% to any of the sequences shown in SEQ ID NOS: 3, 4 or 5,respectively, in WO 99/19467.

The Bacillus alpha-amylase may also be a variant and/or hybrid,especially one described in any of WO 96/23873, WO 96/23874, WO97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents arehereby incorporated by reference). Specific alpha-amylase variants aredisclosed in U.S. Pat. Nos. 6,093,562, 6,187,576, and 6,297,038 (herebyincorporated by reference) and include Bacillus stearothermophilusalpha-amylase (BSG alpha-amylase) variants having a deletion of one ortwo amino acids at positions R179 to G182, preferably a double deletiondisclosed in WO 96/23873—see, e.g., page 20, lines 1-10 (herebyincorporated by reference), preferably corresponding to delta (181-182)compared to the amino acid sequence of Bacillus stearothermophilusalpha-amylase set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or thedeletion of amino acids R179 and G180 using SEQ ID NO: 3 in WO 99/19467for numbering (which reference is hereby incorporated by reference).Even more preferred are Bacillus alpha-amylases, especially Bacillusstearothermophilus alpha-amylases, which have a double deletioncorresponding to delta (181-182) and further comprise a N193Fsubstitution (also denoted I181*+G182*+N193F) compared to the wild-typeBSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 3disclosed in WO 99/19467. The bacterial alpha-amylase may also have asubstitution in a position corresponding to S239 in the Bacilluslicheniformis alpha-amylase shown in SEQ ID NO: 4 in WO 99/19467, or aS242 variant of the Bacillus stearothermophilus alpha-amylase of SEQ IDNO: 3 in WO 99/19467. In a preferred embodiment the alpha-amylase isselected from the group of Bacillus stearomthermphilus alpha-amylasevariants:

I181*+G182*+N193F+E129V+K177L+R179E;I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+Q254S+M284V; and

181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S (using SEQ IDNO: 3 disclosed in WO 99/19467 for numbering).

Bacterial Hybrid Alpha-Amylases

The alpha-amylase may be a hybrid alpha-amylase, e.g., an alpha-amylasecomprising 445 C-terminal amino acid residues of the Bacilluslicheniformis alpha-amylase (shown in SEQ ID NO: 4 of WO 99/19467) andthe 37 N-terminal amino acid residues of the alpha-amylase derived fromBacillus amyloliquefaciens (shown in SEQ ID NO: 5 of WO 99/19467), withone or more, especially all, of the following substitutions:G48A+T491+G107A+H156Y+A181T+N190F+1201F+A209V+Q264S (using the Bacilluslicheniformis numbering in SEQ ID NO: 4 of WO 99/19467). Also preferredare variants having one or more of the following mutations (orcorresponding mutations in other Bacillus alpha-amylases): H154Y, A181T,N190F, A209V and Q264S and/or the deletion of two residues betweenpositions 176 and 179, preferably the deletion of E178 and G179 (usingSEQ ID NO: 5 of WO 99/19467 for position numbering).

Fungal Alpha-Amylases

Fungal alpha-amylases include alpha-amylases derived from a strain ofAspergillus, such as, Aspergillus kawachii, Aspergillus niger andAspergillus oryzae alpha-amylases.

A preferred acidic fungal alpha-amylase is an alpha-amylase whichexhibits a high identity, i.e., at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99% or even 100% identity to the mature partof the amino acid sequence shown in SEQ ID NO: 10 in WO 96/23874.

Another preferred acid alpha-amylase is derived from a strain ofAspergillus niger. In a preferred embodiment the acid fungalalpha-amylase is an Aspergillus niger alpha-amylase disclosed as“AMYA_ASPNG” in the Swiss-prot/TeEMBL database under the primaryaccession no. P56271 and described in WO 89/01969 (Example3—incorporated by reference).

Other wild-type alpha-amylases include those derived from a strain ofMeripilus and Rhizomucor, preferably a strain of Meripilus giganteus orRhizomucor pusillus (WO 2004/055178 which is incorporated herein byreference).

In a preferred embodiment, the alpha-amylase is derived from Aspergilluskawachii (Kaneko et al., 1996, J. Ferment. Bioeng. 81: 292-298,“Molecular-cloning and determination of the nucleotide-sequence of agene encoding an acid-stable alpha-amylase from Aspergillus kawachii”;and further as EMBL: #AB008370).

The fungal alpha-amylase may also be a wild-type enzyme comprising astarch-binding domain (SBD) and an alpha-amylase catalytic domain, or avariant thereof.

Fungal Hybrid Alpha-Amylases

In a preferred embodiment, the fungal acid alpha-amylase is a hybridalpha-amylase. Examples of fungal hybrid alpha-amylases include the onesdisclosed in WO 2005/003311, U.S. Patent Application Publication No.2005/0054071 (Novozymes), and WO 2006/069290 (Novozymes), which arehereby incorporated by reference. A hybrid alpha-amylase may comprise analpha-amylase catalytic domain (CD) and a carbohydrate-bindingdomain/module (CBM), such as a starch binding domain (SBD), andoptionally a linker.

Examples of hybrid alpha-amylases include those disclosed in Tables 1 to5 of the examples in WO 2006/069290 including the variant with thecatalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO: 100 in WO2006/069290), Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMGlinker and SBD (SEQ ID NO: 101 in WO 2006/069290), Rhizomucor pusillusalpha-amylase with Aspergillus niger glucoamylase linker and SBD (whichis disclosed in Table 5 as a combination of amino acid sequences SEQ IDNO: 20, SEQ ID NO: 72 and SEQ ID NO: 96 in U.S. application Ser. No.11/316,535) or as V039 in Table 5 in WO 2006/069290, and Meripilusgiganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD(SEQ ID NO: 102 in WO 2006/069290). Other hybrid alpha-amylases arelisted in Tables 3, 4, 5, and 6 in Example 4 in U.S. application Ser.No. 11/316,535 and WO 2006/069290 (which are hereby incorporated byreference).

In a preferred embodiment the alpha-amylase is an alpha-amylase derivedfrom Rhizomucor pusillus with an Aspergillus niger glucoamylase linkerand starch-binding domain (SBD), preferably the one shown in SEQ ID NO:7 in WO2013/006756, preferably having one or more of the followingsubstitutions: G128D, D143N, especially G128D+D143N.

Other examples of hybrid alpha-amylases include those disclosed in U.S.Patent Application Publication No. 2005/0054071, including thosedisclosed in Table 3 on page 15, such as Aspergillus niger alpha-amylasewith Aspergillus kawachii linker and starch binding domain.

Other alpha-amylases exhibit a high degree of sequence identity to anyof above mentioned alpha-amylases, i.e., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or even 100% identity to themature enzyme sequences disclosed above.

Commercial Alpha-Amylase Products

Preferred commercial compositions comprising alpha-amylase includeMYCOLASE™ (DSM), BAN™, TERMAMYL™ SC, FUNGAMYL™, LIQUOZYME™ X, LIQUOZYME™SC and SAN™ SUPER, SAN™ EXTRA L (Novozymes A/S) and CLARASE™ L-40,000,DEX-LO™, SPEZYME™ FRED, SPEZYME™ AA, SPEZYME™ ALPHA, SPEZYME™ DELTA AA,GC358, GC980, SPEZYME™ CL and SPEZYME™ RSL (DuPont IndustrialBiosciences), and the acid fungal alpha-amylase from Aspergillus nigerreferred to as SP288 (available from Novozymes A/S, Denmark).

Carbohydrate-Source Generating Enzymes (Saccharifying Enzymes)

The term “carbohydrate-source generating enzyme” includes glucoamylase(a glucose generator), beta-amylase and maltogenic amylase (both maltosegenerators) and also alpha-glucosidase, isoamylase and pullulanase. Acarbohydrate-source generating enzyme is capable of producing acarbohydrate that can be used as an energy-source by the fermentingorganism(s) in question, for instance, when used in a process of theinvention for producing a fermentation product, such as ethanol. Thegenerated carbohydrate may be converted directly or indirectly to thedesired fermentation product, preferably ethanol. A mixture ofcarbohydrate-source generating enzymes may be used. Blends includemixtures comprising at least a glucoamylase and an alpha-amylase,especially an acid amylase, even more preferred an acid fungalalpha-amylase.

In a conventional starch-to-ethanol process (i.e., including aliquefaction step), the ratio may preferably be as defined in EP 140410,especially when saccharification and fermentation are carried outsimultaneously.

Glucoamylases

The term “glucoamylase” (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3)is an enzyme, which catalyzes the release of D-glucose from thenon-reducing ends of starch or related oligo- and polysaccharidemolecules.

The glucoamylase may added in an amount of 0.001 to 10 AGU/g DS,preferably from 0.01 to 5 AGU/g DS, such as around 0.1, 0.3, 0.5, 1 or 2AGU/g DS, especially 0.1 to 0.5 AGU/g DS or 0.02-20 AGU/g DS, preferably0.1-10 AGU/g DS.

A glucoamylase may be derived from any suitable source, e.g., derivedfrom a microorganism or a plant. Preferred glucoamylases are of fungalor bacterial origin, selected from the group consisting of Aspergillusglucoamylases, in particular Aspergillus niger G1 or G2 glucoamylase(Boel et al., 1984, EMBO J. 3(5): 1097-1102), or variants thereof, suchas those disclosed in WO 92/00381, WO 00/04136 and WO 01/04273 (fromNovozymes, Denmark); the A. awamori glucoamylase disclosed in WO84/02921, Aspergillus oryzae glucoamylase (Hata et al., 1991, Agric.Biol. Chem. 55(4): 941-949), or variants or fragments thereof. OtherAspergillus glucoamylase variants include variants with enhanced thermalstability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505);D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chenet al., 1994, Biochem. J. 301: 275-281); disulphide bonds, A246C(Fierobe et al., 1996, Biochemistry 35: 8698-8704; and introduction ofPro residues in positions A435 and S436 (Li et al., 1997, Prot. Eng. 10:1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 andNagasaka et al., 1998, Appl. Microbiol. Biotechnol. 50: 323-330),Talaromyces glucoamylases, in particular derived from Talaromycesduponti, Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus(U.S. Patent No. Re. 32, 153), and Talaromyces thermophilus (U.S. Pat.No. 4,587,215). Gloeophyllum sp. (US 2012/0214196).

In a specific embodiment the glucoamylase is from a strain of the genusPenicillium, especially a strain of Penicillium oxalicum, in particularthe Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO2011/127802. In a preferred embodiment the glucoamylase is a variant ofthe Penicillium oxalicum glucoamylase disclosed as SEQ ID NO: 2 in WO2011/127802 having a K79V substitution using the mature polypeptide(amino acids 22-616 of SEQ ID NO: 2) for numbering, and described in WO2013/036526. In a preferred embodiment the glucoamylase is a variant ofthe Penicillium oxalicum glucoamylase disclosed as amino acids 22-616 ofSEQ ID NO: 2 in WO 2011/127802 having a K79V substitution and one ormore of the following substitutions P2N, P4S, P11F, T65A, Q327F,especially P2N+P4S+P11F+T65A+Q327F as described in WO2013/053801.

In a specific embodiment the glucoamylase is from a strain of the genusPycnoporus, especially a strain of Pycnoporus sanguineus, in particularthe Pycnoporus sanguineus glucoamylase disclosed as SEQ ID NO: 2, 4, or6 in WO 2011/066576. In a preferred embodiment the enzyme compositioncomprises the glucoamylase shown as amino acids 19-573 of SEQ ID NO: 6in WO 2011/066576.

In a specific embodiment the glucoamylase is from a strain of the genusGloeophillum, especially a strain of Gloeophyllum trabeum, in particularthe Gloeophyllum trabeum glucoamylase disclosed as SEQ ID NO: 18 in WO2011/068803. In an especially preferred embodiment the enzymecomposition comprises the Gloeophyllum trabeum glucoamylase shown inamino acids 18-576 of SEQ ID NO: 18 in WO2011/068803, and having one ormore of the following substitutions: S95P, A121P, especially S95P+A121Pusing the mature polypeptide (positions 18-576 of SEQ ID NO: 18) fornumbering.

In a specific embodiment the glucoamylase is from a strain of the genusGloeophillum, especially a strain of Gloeophillum sepiarium, inparticular the mature Gloeophillum sepiarium glucoamylase disclosed asamino acids 18-573 of SEQ ID NO: 2 in WO2011/068803.

Bacterial glucoamylases include glucoamylases from Clostridium, inparticular C. thermoamylolyticum (EP 135138) and C.thermohydrosulfuricum (WO 86/01831), Trametes cingulata, Pachykytosporapapyracea, and Leucopaxillus giganteus, all disclosed in WO 2006/069289;or Peniophora rufomarginata disclosed in PCT/US2007/066618; or a mixturethereof. A hybrid glucoamylase may be used in the present invention.Examples of hybrid glucoamylases are disclosed in WO 2005/045018.Specific examples include the hybrid glucoamylase disclosed in Tables 1and 4 of Example 1 (which hybrids are hereby incorporated by reference).

The glucoamylase may have a high degree of sequence identity to any ofabove mentioned glucoamylases, i.e., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or even 100% identity to themature enzymes sequences mentioned above.

Commercially available glucoamylase compositions include AMG 200L; AMG300L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL,SPIRIZYME™ B4U, SPIRIZYME ULTRA™ and AMG™ E (from Novozymes A/S,Denmark); OPTIDEX™ 300, GC480™ and GC147™ (from DuPont IndustrialBiosciences, USA); AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900,G-ZYME™ and G990 ZR (from DuPont Industrial Biosciences).

Glucoamylases may be added in an amount of 0.02-20 AGU/g DS, preferably0.1-10 AGU/g DS, especially between 1-5 AGU/g DS, such as 0.1-2 AGU/gDS, such as 0.5 AGU/g DS or in an amount of 0.0001-20 AGU/g DS,preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/g DS, suchas 0.1-2 AGU/g DS.

Beta-Amylases

A beta-amylase (E.C 3.2.1.2) is the name traditionally given toexo-acting maltogenic amylases, which catalyze the hydrolysis of1,4-alpha-glucosidic linkages in amylose, amylopectin and relatedglucose polymers. Maltose units are successively removed from thenon-reducing chain ends in a step-wise manner until the molecule isdegraded or, in the case of amylopectin, until a branch point isreached. The maltose released has the beta anomeric configuration, hencethe name beta-amylase.

Beta-amylases have been isolated from various plants and microorganisms(Fogarty and Kelly, 1979, Progress in Industrial Microbiology 15:112-115). These beta-amylases are characterized by having a temperatureoptimum in the range from 40° C. to 65° C. and a pH optimum in the rangefrom 4.5 to 7. A commercially available beta-amylase from barley isNOVOZYM™ WBA from Novozymes A/S, Denmark and SPEZYME™ BBA 1500 fromDuPont Industrial Biosciences, USA.

Maltogenic Amylases

The amylase may also be a maltogenic alpha-amylase (glucan1,4-alpha-maltohydrolase, EC 3.2.1.133), which catalyzes the hydrolysisof amylose and amylopectin to maltose in the alpha-configuration. Amaltogenic amylase from Bacillus stearothermophilus strain NCIB 11837 iscommercially available from Novozymes A/S. Maltogenic alpha-amylases aredescribed in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, whichare hereby incorporated by reference. The maltogenic amylase may beadded in an amount of 0.05-5 mg total protein/gram DS or 0.05-5 MANU/gDS.

Pullulanases

Pullulanases (E.C. 3.2.1.41, pullulan 6-glucano-hydrolase), aredebranching enzymes characterized by their ability to hydrolyze thealpha-1,6-glycosidic bonds in, for example, amylopectin and pullulan.

The pullulanase according to the invention, and in addition any furtherpullulanase may be added, preferably a bacterial pullulanase, preferablyderived from a strain of the genus Bacillus, especially derived from astrain of Bacillus deramificans, Bacillus subtilis, Bacillusamyloderamificans, or Bacillus acidopullulyticus.

The pullulanase may according to the invention be added in an effectiveamount which include the preferred range of from between 1-100 micro gper g DS, especially from 10-60 micro g per g DS. Pullulanase activitymay be determined as NPUN. An Assay for determination of NPUN isdescribed in the “Materials & Methods”-section below.

In a preferred embodiment, the pullulanase is used in an amount between1-100 micro g enzyme protein per g DS, preferably between 10-60 micro genzyme protein per g DS.

Suitable commercially available pullulanase products include PROMOZYMED, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-1000, OPTIMAX L-300(DuPont Industrial Biosciences), and AMANO 8 (Amano, Japan).

The present invention is further described by the following numberedembodiments:

Embodiment 1

A pullulanase variant comprising a substitution at one or more positionscorresponding to positions 393, 143, 150, 243, 244, 346, 368, 370, 373,381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 631, 632, 665 and 699of the polypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity; and

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orb) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orc) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ord) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ore) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Embodiment 2

The pullulanase variant according to embodiment 1, comprising asubstitution at two or more positions corresponding to positions 393,143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430,456, 486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQID NO: 3 wherein the variant has pullulanase activity; and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orb) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orc) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ord) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ore) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Embodiment 3

The pullulanase variant according to embodiment 1, comprising asubstitution at three or more positions corresponding to positions 393,143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430,431, 456, 486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptideof SEQ ID NO: 3 wherein the variant has pullulanase activity; and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orb) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orc) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ord) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ore) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Embodiment 4

The pullulanase variant according to embodiment 1, comprising asubstitution at four or more positions corresponding to positions 393,143, 150, 243, 244, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430,431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 of thepolypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity; and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orb) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orc) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ord) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ore) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Embodiment 5

The pullulanase variant according to embodiment 1, comprising asubstitution at five or more positions corresponding to positions 393,143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 385, 387, 402, 429,430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 of thepolypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity; and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orb) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orc) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ord) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ore) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Embodiment 6

The pullulanase variant according to embodiment 1, comprising asubstitution at six or more positions corresponding to positions 393,143, 150, 243, 244, 345, 346, 368, 370, 373, 381, 382, 385, 387, 402,429, 430, 431, 432, 456, 486, 492, 610, 624, 631, 632, 665 and 699 ofthe polypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity; and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 3, particularly the variant has at least 60% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 65%, more particularly at least 70%, moreparticularly at least 60%, more particularly at least 75%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orb) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 6, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; orc) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ord) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 16, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; ore) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 17, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%.

Embodiment 7

The pullulanase variant according to any of the preceding embodiments,comprising a substitution at one position corresponding to positions393, 143, 150, 243, 244, 345, 346, 368, 486, 492, 610, 624, or 699 ofthe polypeptide of SEQ ID NO: 3 wherein the variant has pullulanaseactivity.

Embodiment 8

The variant of any of embodiments 1-7, wherein the number ofsubstitutions is 1-20, e.g., 1-10 and 1-5, such as 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 substitutions.

Embodiment 9

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 393.

Embodiment 10

The variant of embodiment 09, wherein the substitution is with Alanine.

Embodiment 11

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 143.

Embodiment 12

The variant of embodiment 11, wherein the substitution is with Valine.

Embodiment 13

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 150.

Embodiment 14

The variant of embodiment 13, wherein the substitution is with Arginine.

Embodiment 15

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 243.

Embodiment 16

The variant of embodiment 15, wherein the substitution is with GlutamicAcid.

Embodiment 17

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 244.

Embodiment 18

The variant of embodiment 17, wherein the substitution is with Lysine.

Embodiment 19

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 345.

Embodiment 20

The variant of embodiment 19, wherein the substitution is with Proline.

Embodiment 21

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 346.

Embodiment 22

The variant of embodiment 21, wherein the substitution is with Serine.

Embodiment 23

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 368.

Embodiment 24

The variant of embodiment 23, wherein the substitution is with GlutamicAcid.

Embodiment 25

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 370.

Embodiment 26

The variant of embodiment 25, wherein the substitution is with Serine.

Embodiment 27

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 373.

Embodiment 28

The variant of embodiment 27, wherein the substitution is with Leucine.

Embodiment 29

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 381.

Embodiment 30

The variant of embodiment 29, wherein the substitution is with Valine.

Embodiment 31

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 382.

Embodiment 32

The variant of embodiment 31, wherein the substitution is withThreonine.

Embodiment 33

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 385.

Embodiment 34

The variant of embodiment 33, wherein the substitution is with GlutamicAcid.

Embodiment 35

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 402.

Embodiment 36

The variant of embodiment 35, wherein the substitution is withThreonine.

Embodiment 37

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 429.

Embodiment 38

The variant of embodiment 37, wherein the substitution is with Valine.

Embodiment 39

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 430.

Embodiment 40

The variant of embodiment 39, wherein the substitution is with Arginine.

Embodiment 41

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 431.

Embodiment 42

The variant of embodiment 41, wherein the substitution is with GlutamicAcid.

Embodiment 43

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 432.

Embodiment 44

The variant of embodiment 43, wherein the substitution is withPhenylalanine.

Embodiment 45

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 456.

Embodiment 46

The variant of embodiment 45, wherein the substitution is with Alanine.

Embodiment 47

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 486.

Embodiment 48

The variant of embodiment 47, wherein the substitution is with Cysteine.

Embodiment 49

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 492.

Embodiment 50

The variant of embodiment 49, wherein the substitution is with Serine orAlanine.

Embodiment 51

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 610.

Embodiment 52

The variant of embodiment 51 wherein the substitution is with Leucine orArginine.

Embodiment 53

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 624.

Embodiment 54

The variant of embodiment 53, wherein the substitution is with Serine.

Embodiment 55

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 631.

Embodiment 56

The variant of embodiment 55 wherein the substitution is with Serine.

Embodiment 57

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 632.

Embodiment 58

The variant of embodiment 57 wherein the substitution is with Cysteine.

Embodiment 59

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 665.

Embodiment 60

The variant of embodiment 59 wherein the substitution is withIsoleucine.

Embodiment 61

The variant of any of embodiments 1-8, which comprises a substitution ata position corresponding to position 699.

Embodiment 62

The variant of embodiment 61 wherein the substitution is with Arginine.

Embodiment 63

The variant according to embodiment 1-62, wherein the variant comprisesor consists of one or more substitutions selected from the groupconsisting of 393A, 143G, 150R, 243E, 244K, 345P, 346S, 368G, 370S,373L, 381V, 382T, 385F, 387L, 402T, 429V, 430R, 431E, 432F, 456A, 486C,492S, 610R,L, 624S, 631S, 632C, 665I and 699R.

The variant of any of the preceding embodiments, wherein the variantcomprises at least one of the following substitutions or combinations ofsubstitutions:

N368G; E699R; E150R; N346S; N243E; S244K; V143G; N393A; N610R; N610L;G624S; F456A; T492S; V486C+T492S; N368G+M402T; T631S+S632C;V486C+T492S+T631S+S632C; N393A+T631S+S632C; T631S+S632C+E699R;N393A+V486C+T492S+T631S+S632C; N393A+G624S+S632C;N393A+N610R+T631S+S632C; N393A+G624S+T631S+S632C;N393A+N610R+G624S+T631S+S632C; N393A+V486C+T492S+G624S+T631S+S632C;N393A+V486C+T492S+N610R+G624S+T631S+S632C;N368G+N393A+V486C+T492S+N610R+G624S+T631S+S632C;N393A+V486C+T492S+N610R+G624S+T631S+S632C+E699R;N346S+N393A+V486C+T492S+N610R+G624S+T631S+S632C;N393A+F456A+V486C+T492S+N610R+G624S+T631S+S632C;N393A+T492S+N610R+G624S+T631S+S632C;N368G+N393A+T492S+N610R+G624S+T631S+S632C;A345P+N393A+V486C+T492S+N610R+G624S+T631S+S632C;N368G+K370S+I373L+N393A+V486C+T492S+N610R+G624S+T631S+S632C;I381V+Q385E+Q387L+N393A+V486C+T492S+N610R+G624S+T631S+S632C;I381V+N382T+Q385E+Q387L+N393A+V486C+T492S+N610R+G624S+T631S+S632C;A345P+N368G+N393A+T492S+N610R+G624S+T631S+S632C;N368G+I381V+Q385E+Q387L+N393A+T492S+N610R+G624S+T631S+S632C;A345P+N368G+I381V+Q385E+Q387L+N393A+T492S+N610R+G624S+T631S+S632C;A345P+N368G+I381V+Q385E+Q387L+N393A+T492S+N610R+G624S+T631S+S632C+V665I;N393A+T430R+Q431E+L432F+V486C+T492S+N610R+G624S+T631S+S632C;N393A+Q431E+L432F+V486C+T492S+N610R+G624S+T631S+S632C;N393A+I429V+Q431E+V486C+T492S+N610R+G624S+T631S+S632C;N393A+I429V+T430R+Q431E+L432F+V486C+T492S+N610R+G624S+T631S+S632C;N368G+N393A+A492S,A; N368G+N393A; N393A+N610R; N368G+N393A+N610R;N368G+N393A+T492S,A+N610R+G624S; N368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F; N368G+N393A+N610R+G624S+T631S+S632C;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+N610R+G624S+T631S+S632C+Q431E+L432F. Embodiment 64

A variant catalytic domain, wherein the variant catalytic domaincomprises a substitution at one or more positions corresponding topositions 393, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456,486, 492, 610, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3;and;

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3;orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6;orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 9;ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

Embodiment 65

The variant catalytic domain according to embodiment 64 comprising asubstitution at two or more positions corresponding to positions 393,346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610,624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3 whereinthe variant catalytic domain has pullulanase activity, and;

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3;orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6;orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 9;ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

Embodiment 66

The variant catalytic domain according to embodiment 64 comprising asubstitution at three or more positions corresponding to positions 393,346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 456, 486, 492,610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3wherein the variant catalytic domain has pullulanase activity, and;

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3;orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6;orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 9;ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

Embodiment 67

The variant catalytic domain according to embodiment 64 comprising asubstitution at four or more positions corresponding to positions 393,346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456, 486,492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3wherein the variant catalytic domain has pullulanase activity, and;

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3;orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6;orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 9,ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

Embodiment 68

The variant catalytic domain according to embodiment 64 comprising asubstitution at five or more positions corresponding to positions 393,345, 346, 368, 370, 373, 381, 385, 387, 402, 429, 430, 431, 432, 456,486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ IDNO: 3 wherein the variant catalytic domain has pullulanase activity;and;

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3;orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6;orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 9;ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

Embodiment 69

The variant catalytic domain according to embodiment 64 comprising asubstitution at six or more positions corresponding to positions 393,345, 346, 368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432,456, 486, 492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQID NO: 3 wherein the variant catalytic domain has pullulanase activity;and;

a) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 3;orb) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 6;orc) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 9;ord) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 16; ore) wherein the variant catalytic domain has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 430 to 928 of SEQ IDNO: 17.

Embodiment 70

A pullulanase variant comprising the variant catalytic domain accordingto any of embodiments 64 to 69, wherein the pullulanase variant haspullulanase activity and increased thermoactivity compared to the parentpullulanase, and the variant has at least 60% relative activity whenmeasured at 70° C. relative to activity at 65° C., more particularly atleast 65%, more particularly at least 70%, more particularly at least60%, more particularly at least 75%, more particularly at least 80%,more particularly at least 90%, more particularly at least 100%.

Embodiment 71

The pullulanase variant according to embodiment 70, wherein the variantcomprises or consists of one or more substitutions selected from thegroup consisting of 393A, 143G, 150R, 243E, 244K, 345P, 346S, 368G,370S, 373L, 381V, 382T, 385F, 387L, 402T, 429V, 430R, 431E, 432F, 456A,486C, 492S, 610R,L, 624S, 631S, 632C, 665I and 699R.

Embodiment 72

The pullulanase variant according to any of embodiments 64-71, furthercomprising an N-terminal part comprising at least one of the domainsselected from a CBM41 domain, an X45 domain and a CBM48 domain.

Embodiment 73

The pullulanase variant according to embodiment 72, comprising anN-terminal part comprising a CBM41 domain, an X45 domain and a CBM48domain.

Embodiment 74

The pullulanase variant according to embodiment 72-73, furthercomprising a X25 domain.

Embodiment 75

The pullulanase variant according to any of embodiments 64-74, whereinthe variant comprises one or more substitutions selected from the groupconsisting of 393A, 368G, 486C, 492S,A, 610R,L, 624S, 631S, 632C, 431E,432F.

Embodiment 76

The pullulanase variant according to embodiment 75, wherein the variantcomprises at least one of the following substitutions or combinations ofsubstitutions:

368G+393A+492S,A; 368G+393A+492A,S+610R+624S; 393A+492S,A+610R+624S+631S+632C; 368G+393A+492S,A+610R+624S+631S+632C;368G+393A+492S,A+610R+624S+631 S+632C+431E+432F; 368G+393A+610R+624S+631S+632C; or 368G+393A+610R+624S+631S+632C+431E+432F. Embodiment 77

The pullulanase variant according to any of embodiments 1-76, selectedfrom SEQ ID NO: 20 or a pullulanase having at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,or 100% sequence identity to the polypeptide of SEQ ID NO: 20; SEQ IDNO: 21 or a pullulanase having at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99%, or 100%sequence identity to the polypeptide of SEQ ID NO: 21; SEQ ID NO: 22 ora pullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 22; SEQ ID NO: 23 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 23; SEQ ID NO: 24 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 24; SEQ ID NO: 25 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 25; SEQ ID NO: 26 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 26; SEQ ID NO: 27 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 27; SEQ ID NO: 28 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 28.

Embodiment 78

The variants according to embodiments 1-77, which have an improvedproperty relative to the parent, wherein the improved property isincreased thermoactivity.

Embodiment 79

The variant according to embodiments 1-78, wherein the variant hasincreased specific activity toward starch or maltodextrin compared toany of SEQ ID NO: 3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:16, or SEQ IDNO:17.

Embodiment 80

The variants according to embodiment 79, wherein the variant is selectedfrom SEQ ID NO: 20 or a pullulanase having at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,or 100% sequence identity to the polypeptide of SEQ ID NO: 20; SEQ IDNO: 21 or a pullulanase having at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99%, or 100%sequence identity to the polypeptide of SEQ ID NO: 21; SEQ ID NO: 22 ora pullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 22. SEQ ID NO: 23 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 23; SEQ ID NO: 25 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 25; SEQ ID NO: 26 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 26; SEQ ID NO: 27 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 27. SEQ ID NO: 28 or apullulanase having at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99%, or 100% sequenceidentity to the polypeptide of SEQ ID NO: 28.

Embodiment 81

The variants according to any of the preceding embodiments, wherein thevaraints further comprises one of the following substitutions orcombinations of substitutions (using SEQ ID NO: 6 for numbering):

Q258A; Q287R; Q352A; Q356R; Q258A+Q352A+Q356R; Q258A+Q287R+Q352A+Q356R;V212I; H186A; V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+V212I+Q258A+Q287R+Q352A+Q356R; Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q485E+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+Q487L+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+S557A+L559G+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+H421E+Q487L+S557A+L559G+V586A+D686S+E799R.Embodiment 82

The variants according to claim 81, wherein the variants are selectedfrom a pullulanase having at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the polypeptide of SEQ ID NO: 26, and comprising the substitutions(using SEQ ID NO: 6 for numbering):N468G+N493A+N710R+G724S+T731S+S732C+Q531E+L532F, and wherein thevariants further comprises one of the following substitutions orcombinations of substitutions:

Q258A; Q287R; Q352A; Q356R; Q258A+Q352A+Q356R; Q258A+Q287R+Q352A+Q356R;V212I; H186A; V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+V212I+Q258A+Q287R+Q352A+Q356R; Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q485E+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+Q487L+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+S557A+L559G+D686S+E799R;

N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+H421E+Q487L+S557A+L559G+V586A+D686S+E799R;said variants having at least 60% relative activity when measured at 72°C. relative to activity at 65° C. using the PHADEBAS assay.

Embodiment 83

The variants according to embodiment 82, wherein the variants furthercomprises one of the following combinations of substitutions:

N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R.Embodiment 84

The variants according to embodiment 81, wherein the variants areselected from a pullulanase having at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the polypeptide of SEQ ID NO: 27, and comprising thesubstitutions (using SEQ ID NO: 6 for numbering)N468G+N493A+T492S,A+N710R+G724S+T731S+S732C+Q531E+L532F, and wherein thevariants further comprises one of the following substitutions orcombinations of substitutions:

Q258A; Q287R; Q352A; Q356R; Q258A+Q352A+Q356R; Q258A+Q287R+Q352A+Q356R;V212I; H186A; V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+V212I+Q258A+Q287R+Q352A+Q356R; Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;H186A+V212I+Q258A+Q287R+Q352A+Q356R;H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q485E+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+G296R+N322P+Q352A+Q356R+Q487L+V586A+D686S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+C732S+E799R;N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+S557A+L559G+D686S+E799R;

N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+H421E+Q487L+S557A+L559G+V586A+D686S+E799R;said variants having at least 60% relative activity when measured at 72°C. relative to activity at 65° C. using the PHADEBAS assay.

Embodiment 85

The variants according to claim 84, wherein the variants furthercomprise one of the following combinations of substitutions:

N19G+Y27K+H79Y+H186A+V212I+Q258A+Q287R+N322P+Q352A+Q356R+Q487L+D686S+E799R.Embodiment 86

The pullulanase variants according to any of the embodiments 1-80,wherein the variants further comprises one of the followingsubstitutions or combinations of substitutions:

Y27K+H79Y+Q187R+S798R; Y27K+H79Y+Q187R+D586S+S798R;Y27K+H79Y+Q187R+D586S+E699R+S798R; Y27K+H79Y+Q187R+T486S+D586S+S798R;N19G+Y27K+H79Y+Q187R+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q385E+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+Q459G+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+H321E+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+Q459G+D586S+E699R+S798R.Embodiment 87

The variants according to claim 86, wherein the variants are selectedfrom a pullulanase having at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the polypeptide of SEQ ID NO: 28, and comprising the substitutionsN368G+N393A+T492S,A+N610R+G624S+T631 S+S632C+Q431E+L432F, and whereinthe variants further comprises one of the following substitutions orcombinations of substitutions:

Y27K+H79Y+Q187R+S798R; Y27K+H79Y+Q187R+D586S+S798R;Y27K+H79Y+Q187R+D586S+E699R+S798R; Y27K+H79Y+Q187R+T486S+D586S+S798R;N19G+Y27K+H79Y+Q187R+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q385E+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+V196R+Q387L+Q459G+T486C+D586S+C632S+Q675L+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+H321E+Q387L+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+Q459G+D586S+E699R+S798R;N19G+Y27K+H79Y+Q187R+Q387L+D586S+C632S+E699R+S798R;N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+D586S+E699R+S798R;

N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+Q459G+D586S+E699R+S798R, saidvariants having at least 30% relative activity when measured at 76° C.relative to activity at 65° C. using the PHADEBAS assay.

Embodiment 88

The variants according to embodiment 87, wherein the variants furthercomprises one of the following combinations of substitutions:

N19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+D586S+E699R+S798R; orN19G+Y27K+H79Y+Q187R+E310A+D311K+Q387L+Q459G+D586S+E699R+S798R.Embodiment 89

A method for producing a variant pullulanase of a parent pullulanasecomprising substitution of the parent pullulanase at one or morepositions corresponding to positions 393, 143, 150, 243, 244, 345, 346,368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486,492, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQ ID NO: 3wherein the variant has pullulanase activity; and;

a) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 3; orb) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 6; orc) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9; ord) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16; ore) wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 17

Embodiment 90

A variant pullulanase produced by the method of embodiment 89.

Embodiment 91

A composition comprising the variant pullulanase of any of embodiments1-90.

Embodiment 92

The composition according to embodiment 91, comprising one or moreenzymes selected from the group consisting of: glucoamylase,alpha-amylase, beta-amylase, and protease.

Embodiment 93

The composition according to any of embodiments 91 and 92 comprising apullulanase of polypeptide of any of embodiments 1-84 and: i) aglucoamylase, an alpha-amylase and a protease; ii) an alpha-amylase anda protease; iii) a glucoamylase and an alpha-amylase; iv) abeta-amylase; or v) a glucoamylase.

Embodiment 94

A use of a variant pullulanase of any of embodiments 1-90 for productionof a syrup and/or a fermentation product, e.g., ethanol, from a starchcontaining material.

Embodiment 95

The use according to embodiment 94, wherein the starch material isgelatinized or un-gelatinized starch material.

Embodiment 96

A process of producing a fermentation product from starch-containingmaterial comprising the steps of:

(a) liquefying starch-containing material in the presence of an alphaamylase;(b) saccharifying the liquefied material in the presence of aglucoamylase; and(c) fermenting with a fermenting organism; wherein step (a) and/or step(b) is carried out in the presence of a variant pullulanase of any ofembodiments 1-90.

Embodiment 97

A process of producing a fermentation product from starch-containingmaterial, comprising the steps of:

(a) saccharifying starch-containing material at a temperature below theinitial gelatinization temperature of said starch-containing material;and(b) fermenting with a fermenting organismwherein step (a) is carried out using at least a glucoamylase, and avariant pullulanase of any of embodiments 1-90.

Embodiment 98

The process according to embodiment 97, wherein an alpha amylase isadded in step (a).

Embodiment 99

The process according to embodiment 97, wherein saccharification andfermentation is carried out simultaneously.

Embodiment 100

The process according to any of embodiments 96-99, wherein thefermentation product is an alcohol, particularly ethanol.

Embodiment 101

A polynucleotide encoding the variant pullulanase of any of embodiments1-90.

Embodiment 102

A nucleic acid construct comprising the polynucleotide of embodiment101.

Embodiment 103

An expression vector comprising the polynucleotide of embodiment 101.

Embodiment 104

A host cell comprising the polynucleotide of embodiment 101.

Embodiment 105

A method of producing a pullulanase variant of any of embodiments 1-90,comprising cultivating the host cell of embodiment 104 under conditionsconducive for production of the polypeptide.

Embodiment 106

The method of embodiment 105, further comprising recovering thepolypeptide.

Embodiment 107

A whole broth formulation or cell culture composition comprising apolypeptide of any of embodiments 1-90.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

Materials and Methods Determination of Sugar Profile and Solubilised DrySolids

The sugar composition of the starch hydrolysates is determined by HPLCand glucose yield is subsequently calculated as DX. ° BRIX, solubilized(soluble) dry solids of the starch hydrolysates are determined byrefractive index measurement.

EXAMPLES Example 1: Construction of Pullulanase Libraries

Genomic DNAs from Bacillus subtilis strains harboring pullulanase genesfrom Bacillus sp. NCIB11777 (SEQ ID NO: 7 and 8), Bacillus deramificansNN18718 (SEQ ID NO: 4 and 5) and hybrid pullulanase P8, (SEQ ID NO: 1and 2) which is a hybrid enzyme obtained by combining an N-terminalfragment of the pullulanase derived from Bacillus acidopullullyticusdescribed in WO 2009/075682 (SEQ ID NO: 4 in WO2009/075682; GENESEQP:AXB71624), fused to a C-terminal fragment of a pullulanase derived froma Bacillus deramificans strain isolated from a humus sample collected inDenmark (a homologous pullulanase from Bacillus deramificans wasdisclosed in U.S. Pat. No. 6,074,854 and U.S. Pat. No. 5,817,498) wereisolated using NucleoSpin® Tissue kit [MACHEREY-NAGEL]. Pullulanasegenes were amplified from the genomic DNAs using primers having 15 bpoverlaps with an expression vector comprising the genetic elements forBacillus expression and E. coli amplification as described in WO99/43835 using Primer F1 and R1, Primer F2 and R2, and Primer F1 and R2under the following conditions.

Primer F1: (SEQ ID NO: 10)ATGTATTATGGAGCTCTATAAAAATGAGGAGGGAACCGAATGTCCCTAAT ACGTTCTAG Primer R1:(SEQ ID NO: 11) TTATTGATTAACGCGTTTAATTTTGATCAATGACATC Primer F2:(SEQ ID NO: 12) ATGTATTATGGAGCTCTATAAAAATGAGGAGGGAACCGAATGGCTAAAAAACTAATTTATG Primer R2: (SEQ ID NO: 13)TTATTGATTAACGCGTTTACTTTTTACCGTGGTCTG

Phusion polymerase (thermo scientific): Total 20 μl: 1.0 μl Template(100 ng/μl), 4.8 μl H₂O, 4 μl, Phusion HF Buffer:1.6 μl dNTP (2.5 mM):0.2 μl Reverse primer (20 μM), 0.4 μl Phusion (2U/μl), 8.0 μl Forwardmutation primer (1 μM). PCR-program: 98° C./30 sec, 30×(98° C./10 sec,60° C./20 sec, 72° C./3 min), 72° C./5 min, 4° C./∞

The resultant PCR fragments were purified by NucleoSpin® Gel and PCRClean-up kit [MACHEREY-NAGEL] and ligated to the vector by In fusioncloning (Clontech). The in fusion mixture was then introduced into E.coli DH5a, Jet Competent E. coli Cell, BDL. The resultant plasmids wereconfirmed to have designed sequences and named pGMM, pD2 homolog andp008 and used as templates for library construction. All libraries wereconstructed by the following steps. A reverse or forward primer havingNNK or desired mutation(s) at target site(s) with 15 bp overlaps eachother were designed and two PCRs were carried out using Primer1F or 2Fand the reverse primer and the forward primer and Primer 1R or 2R usingappropriate templates (pGMM, pD′ or p008). The resultant PCR fragmentswere purified by NucleoSpin® Gel and PCR Clean-up kit [MACHEREY-NAGEL]and mixed with an expression vector digested with Sacl and MIul and infusion mixture to introduce into E. coli. The resultant E. colilibraries were recovered and transformed into Bacillus subtilis toconstruct Bacillus libraries having desired mutations as shown in Table1a and Table 1b.

TABLE 1a Substitutions of thermostabilized variants of P008 (SEQ ID NO:3) Position Substitution(s) P022 V486C T492S P035 N368G P040 N368G M402TP075 E699R P076 E150R P091 T631S S632C P092 N346S P097 V486C T492S T631SS632C P129 N243E P130 S244K P132 V143G P136 N393A P140 N393A T631S S632CP147 N610R P148 N610L P150 T631S S632C E699R P155 G624S P161 N393A V486CT492S T631S S632C P162 N393A G624S S632C P172 N393A N610R T631S S632CP173 N393A G624S T631S S632C P178 F456A P188 N393A N610R G624S T631SS632C P189 N393A V486C T492S G624S T631S S632C P190 N393A V486C T492SN610R G624S T631S S632C P191 N368G N393A V486C T492S N610R G624S T631SS632C P192 N393A V486C T492S N610R G624S T631S S632C E699R P193 N346SN393A V486C T492S N610R G624S T631S S632C P194 N393A F456A V486C T492SN610R G624S T631S S632C P199 N393A T492S N610R G624S T631S S632C P202N368G N393A T492S N610R G624S T631S S632C P203 A345P N393A V486C T492SN610R G624S T631S S632C P204 N368G K370S I373L N393A V486C T492S N610RG624S T631S S632C P205 I381V Q385E Q387L N393A V486C T492S N610R G624ST631S S632C P206 I381V N382T Q385E Q387L N393A V486C T492S N610R G624ST631S S632C P212 A345P N368G N393A T492S N610R G624S T631S S632C P213N368G I381V Q385E Q387L N393A T492S N610R G624S T631S S632C P222 A345PN368G I381V Q385E Q387L N393A T492S N610R G624S T631S S632C P223 A345PN368G I381V Q385E Q387L N393A T492S N610R G624S T631S S632C V665I P224N393A T430R Q431E L432F V486C T492S N610R G624S T631S S632C P225 N393AQ431E L432F V486C T492S N610R G624S T631S S632C P226 N393A I429V Q431EV486C T492S N610R G624S T631S S632C P227 N393A I429V T430R Q431E L432FV486C T492S N610R G624S T631S S632C P229 N368GI381V Q385E Q387L N393AQ431E L432F T492S N610R G624S T631S S632C P230 A345P N368GI381V Q385EQ387L N393A Q431E L432F T492S N610R G624ST631S S632C P231 A345PN368GI381V Q385E Q387L N393A Q431E L432F T492S N610R G624ST631S S632CV665I P242 A345P N368GI381V Q385E Q387L N393A T492S N610R G624S T631SS632C

Substitutions shown to have thermoactivity improving effects in SEQ IDNO: 3 were tested in SEQ ID NO: 6 according to below tables 1b and ic.Table Ic use the numbering in SEQ ID NO: 3.

TABLE 1b Substitutions of thermostabilized variants of Bacillusderamificans pullulanase (D2 homolog) (SEQ ID NO: 6) Position numberingis according to SEQ ID NO: 6. Variant No. Substitution(s) P233 N468GP234 N493A P235 A592S P236 N710R P237 G724S P238 T731S S732C P239 N710RG724S T731S S732C P240 N468G N493A A592S P241 N468G N493A A592S N710RG724S T731S S732C

TABLE 1c Variants of SEQ ID NO: 6 using numbering according to SEQ IDNO: 3. Variant No. Substitution referring to SEQ ID NO: 3. P233 N368GP234 N393A P235 A492S P236 N610R P237 G624S P240 N368G N393A A492S P259N368G N393A P261 N393A N610R P262 N368G N393A N610R P265 N368G N393AN610R G624S T492A

TABLE 1d Variants of SEQ ID NO: 16 (P258). Variant No. Substitutionreferring to SEQ ID NO: 3. P219 N368G N393A T492A N610R G624S T631SS632C P306 N368G N393A T492A N610R G624S T631S S632C Q431E L432F

TABLE 1e Variants of SEQ ID NO: 17 (P243). Variant No. Substitutionreferring to SEQ ID NO: 3. P252 N368G N393A N610R G624S T631S S632C P303N368G N393A N610R G624S T631S S632C Q431E L432F

In order to test if the improved thermo-activity resulting from theintroduced substitutions in the catalytic domain, could be maintainedwhen N-terminal parts of the parent enzyme were replaced by N-terminalparts from other pullulanases the following hybrid pullulanases wereconstructed.

TABLE 2 Variants having substitutions in the catalytic domain wereconstructed by replacing N-terminal parts with equivalent parts fromother pullulanases. The origin of the different N-terminal domains isshown by SEQ ID and for the CD both the origin and the specificsubstitutions are indicated. CBM41 X45a X25 X45b CBM48 Catalytic Domain,CD P6 SEQ SEQ No SEQ SEQ SEQ ID 6 (or 19) ID 9 ID 9 ID 9 ID 6 P8 SEQ SEQNo SEQ SEQ SEQ ID 6 (or 3) ID 9 ID 9 ID 9 ID 9 (or 3) (or 3) (or 3) (or3) P- SEQ SEQ SEQ SEQ SEQ SEQ ID 6 proD′ ID 6 ID 6 ID 6 ID 6 ID 6 P240SEQ SEQ SEQ SEQ SEQ N368G N393A A492S ID 6 ID 6 ID 6 ID 6 ID 6 (SEQ ID6) P254 SEQ SEQ SEQ SEQ SEQ N368G N393A A492S ID 15 ID 15 ID 15 ID 15 ID6 (SEQ ID 6) P255 SEQ SEQ SEQ SEQ SEQ N368G N393A A492S ID 15 ID 15 ID15 ID 15 ID 9 (SEQ ID 6) P265* SEQ SEQ SEQ SEQ SEQ N368G N393A T492A ID15 ID 15 ID 15 ID 15 ID 9 N610R G624S (SEQ ID 6) P256 SEQ SEQ No SEQ SEQN368G N393A A492S ID 9 ID 9 ID 9 ID 6 (SEQ ID 6) P257 SEQ SEQ No SEQ SEQN368G N393A A492S ID 9 ID 9 ID 9 ID 9 (SEQ ID 6) P267* SEQ SEQ No SEQSEQ N368G N393A T492A ID 9 ID 9 ID 9 ID 6 N610R G624S (SEQ ID 6) P216SEQ SEQ SEQ SEQ SEQ N368G N393A T492S ID 15 ID 15 ID 15 ID 15 ID 9 N610RG624S T631S S632C (SEQ ID 6) P252 SEQ SEQ SEQ SEQ SEQ N368G N393A N610RID 15 ID 15 ID 15 ID 15 ID 9 G624S T631S S632C (SEQ ID 17) P303 SEQ SEQSEQ SEQ SEQ N368G N393A N610R ID 15 ID 15 ID 15 ID 15 ID 9 G624S T631SS632C Q431E L432F (SEQ ID 17) P219 SEQ SEQ SEQ SEQ SEQ N368G N393A T492AID 15 ID 15 ID 15 ID 15 ID 9 N610R G624S T631S S632C (SEQ ID 16) P306SEQ SEQ SEQ SEQ SEQ N368G N393A T492A ID 15 ID 15 ID 15 ID 15 ID 9 N610RG624S T631S S632C Q431E L432F (SEQ ID 16) *P265 and P267 were borne withthe A in position 492, however, this position is contributing to theobserved effect and should not be modified unless the substitution is toS.

Example 2: Construction of Improved Variants of P303

The pullulanase variant, P303, was used as the starting point forfurther variant selection resulting in the below list of specificvariants.

TABLE 3 Substitutions in addition to P303 substitutions (numberingrefers to SEQ ID NO: 26) P316 Q258A P317 Q287R P318 Q352A P319 Q356RP323 Q258A + Q352A + Q356R P324 Q258A + Q287R + Q352A + Q356R P329 V212IP347 H186A P368 V212I + Q258A + Q287R + Q352A + Q356R P369 H186A +V212I + Q258A + Q287R + Q352A + Q356R P370 Y27K + H79Y + V212I + Q258A +Q287R + Q352A + Q356R P372 Q258A + Q287R + N322P + Q352A + Q356R P375H186A + V212I + Q258A + Q287R + N322P + Q352A + Q356R P376 H186A +V212I + Q258A + Q287R + Q352A + Q356R P377 H186A + V212I + Q258A +Q287R + N322P + Q352A + Q356R P378 Y27K + H79Y + H186A + V212I + Q258A +Q287R + Q352A + Q356R P385 Y27K + H79Y + H186A + V212I + Q258A + Q287R +N322P + Q352A + Q356R P397 Y27K + H79Y + H186A + V212I + Q258A + Q287R +N322P + Q352A + Q356R + D686S P415 N19G + Y27K + H79Y + H186A + V212I +Q258A + Q287R + N322P + Q352A + Q356R + D686S P416 N19G + Y27K + H79Y +H186A + V212I + Q258A + Q287R + G296R + N322P + Q352A + Q356R + D686SP417 Y27K + H79Y + H186A + V212I + Q258A + Q287R + N322P + Q352A +Q356R + V586A + D686S P418 Y27K + H79Y + H186A + V212I + Q258A + Q287R +N322P + Q352A + Q356R + D686S + E799R P424 N19G + Y27K + H79Y + H186A +V212I + Q258A + Q287R + N322P + Q352A + Q356R + V586A + D686S P425N19G + Y27K + H79Y + H186A + V212I + Q258A + Q287R + N322P + Q352A +Q356R + D686S + E799R P426 N19G + Y27K + H79Y + H186A + V212I + Q258A +Q287R + N322P + Q352A + Q356R + Q485E + D686S + E799R P427 N19G + Y27K +H79Y + H186A + V212I + Q258A + Q287R + N322P + Q352A + Q356R + Q487L +D686S + E799R P428 N19G + Y27K + H79Y + H186A + V212I + Q258A + Q287R +N322P + Q352A + Q356R + V586A + D686S + E799R P435 N19G + Y27K + H79Y +H186A + V212I + Q258A + Q287R + G296R + N322P + Q352A + Q356R + V586A +D686S + E799R P436 N19G + Y27K + H79Y + H186A + V212I + Q258A + Q287R +G296R + N322P + Q352A + Q356R + Q487L + V586A + D686S + E799R P472N19G + Y27K + H79Y + H186A + V212I + Q258A + Q287R + N322P + Q352A +Q356R + Q487L + D686S + C732S + E799R P484 N19G + Y27K + H79Y + H186A +V212I + Q258A + Q287R + N322P + Q352A + Q356R + Q487L + D686S + C732S +E799R P485 N19G + Y27K + H79Y + H186A + V212I + Q258A + Q287R + N322P +Q352A + Q356R + Q487L + S557A + L559G + D686S + E799R P486 N19G + Y27K +H79Y + H186A + V212I + Q258A + Q287R + N322P + Q352A + Q356R + H421E +Q487L + S557A + L559G + V586A + D686S + E799R

Example 3: Construction of Variants P287 and P380

Starting from P202, described in example 1 above, three furthersubstitutions, N222P+Q252A+Q256R, were introduced in order to generateP287.

Thus, P287, is SEQ ID NO: 3 comprising the substitutions:N222P+Q252A+Q256R+N368G+N393A+T492S+N610R+G624S+T631S+S632C.

Another variant, P380, was generated by replacing the catalytic domainof P008 with the catalytic domain from P306. P380 is included herein asSEQ ID NO: 28.

Thus, P380, has the structure as shown below:

CBM41 X45a X25 X45b CBM48 Catalytic Domain, CD P380 SEQ SEQ No SEQ SEQN368G N393A T492A ID 9 ID 9 ID 9 ID 9 N610R G624S T631S (or 3) (or 3)(or 3) (or 3) S632C Q431E L432F (SEQ ID 16)

Example 4: Construction of Improved Variants of P380

The pullulanase variant, P380, was used as the starting point forfurther variant selection resulting in the below list of specificvariants.

TABLE 4 Substitution in addition to P380 substitutions (numbering refersto SEQ ID NO: 3) P388 Y27K + H79Y + Q187R + S798R P398 Y27K + H79Y +Q187R + D586S + S798R P403 Y27K + H79Y + Q187R + D586S + E699R + S798RP408 Y27K + H79Y + Q187R + T486S + D586S + S798R P423 N19G + Y27K +H79Y + Q187R + T486C + D586S + E699R + S798R P430 N19G + Y27K + H79Y +Q187R + Q385E + T486C + D586S + E699R + S798R P431 N19G + Y27K + H79Y +Q187R + Q387L + T486C + D586S + E699R + S798R P443 N19G + Y27K + H79Y +Q187R + Q387L + Q459G + T486C + D586S + C632S + E699R + S798R P444N19G + Y27K + H79Y + Q187R + Q387L + T486C + D586S + C632S + Q675L +E699R + S798R P448 N19G + Y27K + H79Y + Q187R + V196R + Q387L + T486C +D586S + C632S + E699R + S798R P449 N19G + Y27K + H79Y + Q187R + V196R +Q387L + T486C + D586S + C632S + Q675L + E699R + S798R P450 N19G + Y27K +H79Y + Q187R + V196R + Q387L + Q459G + T486C + D586S + C632S + Q675L +E699R + S798R P470 N19G + Y27K + H79Y + Q187R + Q387L + D586S + E699R +S798R P481 N19G + Y27K + H79Y + Q187R + H321E + Q387L + D586S + E699R +S798R P482 N19G + Y27K + H79Y + Q187R + Q387L + Q459G + D586S + E699R +S798R P483 N19G + Y27K + H79Y + Q187R + Q387L + D586S + C632S + E699R +S798R P493 N19G + Y27K + H79Y + Q187R + E310A + D311K + Q387L + D586S +E699R + S798R P507 N19G + Y27K + H79Y + Q187R + E310A + D311K + Q387L +Q459G + D586S + E699R + S798R

Example 5: Pullulanase Assay Red-Pullulan Assay (Meqazyme) SubstrateSolution

0.1 g red-pullulan (megazyme S-RPUL)0.75 ml 2M sodium acetate, pH5.5

14.25 ml H2O

10 μl of enzyme samples were mixed with 80 μl of substrate solution andincubated at set temperatures (ex. 55, 60, 650C) for 20 min. 50 μl ofethanol was added to the reaction mixtures and centrifuge for 10 min. at3500 rpm.

The supernatants were carefully taken out and the absorbance, A510 wasdetermined.

PAHBAH-Pullulan Assay Substrate Solution

0.15 g BH4-pullulan25 ml 50 mM Na acetate buffer, pH5.5PAHBAH solution0.0552 g Bismuth (III)-acetate

0.2 g PAHBAH

0.5 g Potassium sodium tartrate, tetrahydrate

10 ml 500 mM NaOH

10 μl of enzyme samples were mixed with 110 μl of substrate solution andincubated at set temperatures (e.g., 55, 60, 650C) for 20 min. 40 μl ofPAHBAH solution was added to the reaction mixtures, incubated foranother 20 min at 50° C. and the absorbance, A405 was determined.

Lintner Soluble Waxy Starch Assay Substrate Solution

0.2 g Lintner's waxy corn starch2.5 ml 2M sodium acetate

97.5 ml H₂O

5 μl of enzyme samples were mixed with 100 μl of substrate solution andincubated at set temperatures (e.g., 55, 60, 65, 70, 750C) for 20 min.100 μl of 0.15% I₂/1.5% KI solution was added to the reaction mixturesand the absorbance, A610 was determined.

PHADEBAS Assay Substrate Soln.

1 tablet of PHADEBAS alpha-amylase tablet

5 ml 50 mM Na acetate buffer, pH5

40 sec. microwave oven up to boiling

Stop Soln.

18% acetic acid

Assay Method

Enzyme Reaction in 96 Well PCR Tube

10 ul of enzyme samples were mixed with 100 ul of substrate solution andincubated at set temperatures (eg. 55, 60, 650C) for 20 min. 50 ul ofstop solution was added to the reaction mixtures and centrifuge for 10minutes at 3500 rpm. The supernatants were carefully taken out and theabsorbance at A600 was read.

Example 6: Evaluation of Thermoactivity

Bacillus libraries constructed as in example 1 were fermented in 96 wellMTPs containing TB-gly medium (13.3 g/L Bacto™ Tryptone, 26.6 g/L Bacto™Yeast extract D, 4.4 g/L Glycerol) with 6 mg/L chloramphenicol at 220rpm, 370C and pullulanase activities were measured at severaltemperatures by Lintner soluble starch assay and/or Phadebas assaydescribed in EXAMPLE 2. Relative activity of pullulanase variantsshowing higher thermoactivity compared to parental pullulanases as shownin tables.

Relative Relative Relative activity of activity activity of 70° C./ of72° C./ 74° C./ 65° C. (%) 65° C. (%) 65° C. (%) SEQ ID NO: 9 15 6 SEQID NO: 3 54 6 2 P091 98 31 P092 86 4 P129 83 8 P130 71 10 P132 69 11P136 115 61 P147 74 58 P148 64 51 P155 87 14 P178 62 11 P097 118 71 P140106 81 P150 82 44 10 P173 95 75 37

Relative activity of Relative activity of 68° C./60° C. (%) 70° C./60°C. (%) SEQ ID NO: 3 67 35 P022 90 57 P035 113 119 P040 104 105 P075 8672 P076 80 55

Relative activity (%) 70/65 72/65 74/65 76/65 78/65 72/70 74/70 76/70°C. SEQ ID  6%  3%  1% NO: 9 SEQ ID  28%  5%  1% 52%  8% 5% NO: 3 P09172% 12% 6% P136 75%  9% 6% P147 53%  5% 4% P155  59%  6%  0% 59%  8% 6%P140 91% 26% 8% P161 118%  82% 31% 97% 54% 10%  P162 88% 27% 7% P172111%  75% 19% 95% 52% 8% P173 108%  84% 26% 96% 53% 9% P188  97% 100%36% P189 107%  81% 34% P190 122% 106% 61% 13% P191 148% 134% 87% 30%P193 135% 112% 21% P194 124%  68%  9% P199 147% 106% 55%  7% P202 112%68% 12% P203 115% 76% 29% P204 110% 66% 18% P205 103% 63% 42% P206  91%71% 34% P212  97% 84% 22% P213  86% 70% 44% P222  98% 71%  8% P223  92%16%  0% P224 99% 43% 4% P225 108%  76% 8% P226 113%  66% 6% P227 99% 29%3% P230 102% 78% 64% 12%  P231 101% 65% 50% 12%  P242 84% 55% 4%

Relative activity (%) 65° C./60° C. 67° C./60° C. Bacillus deramificanspullulanase 70% 20% (SEQ ID NO: 6) P233 100% 79% P234 102% 81% P235 92%25% P236 100% 64% P240 101% 86%

Relative activity 65° C./60° C. Bacillus deramificans pullulanase (SEQID NO: 6) 65% P261 69% P262 77% P265 84%

Relative activity 70° C./60° C. Bacillus deramificans pullulanase (SEQID NO: 6) 36% P259 106% P240 95%

Example 7: Fermentation of the Bacillus Strains

B. subtilis strains were fermented on a rotary shaking table in 500 mlbaffled flasks containing 100 ml TB-gly with 6 mg/L chloramphenicol at220 rpm, 37° C. The culture was centrifuged (20000×g, 20 min) and thesupernatants were carefully decanted from the precipitates. Thesupernatants were filtered through a 0.45 um filter unit to remove therest of the Bacillus host cells.

Example 8: Purification of Pullulanases

Purification of pullulanases was carried out by β-cyclodextrin affinitycolumn and followed by anion exchange column chromatography. Afterpurification, pullulanases were dialyzed against 20 mM sodium acetatebuffer (pH 5.5) and concentrated.

Example 9: Enzyme Thermostability Measurement

Purified enzyme was diluted with 50 mM sodium acetate pH 5.0 or 4.3 to0.5 mg/ml and mixed with the equal volume of SYPRO Orange (Invitrogen)diluted with Milli-Q water. Thirty microliters of mixture solution wastransfer to LightCycler 480 Multiwell Plate 96 (Roche Diagnostics) andthe plate was sealed.

Equipment parameters of TSA:

-   -   Apparatus: LightCycler 480 Real-Time PCR System (Roche Applied        Science)    -   Scan rate: 0.02° C./sec    -   Scan range: 37-96° C.    -   Scan rate: 1.26° C./min    -   Integration time: 0.5 sec    -   Excitation wave length 465 nm    -   Emission wave length 580 nm

The obtained fluorescence signal was normalized into a range of 0 and 1.The Melting temperature (Tm) was defined as the temperature where thenormalized value is closest to 0.5.

TSA Tm (° C.) pH 4.3 pH 5.0 SEQ ID NO: 9 69 68 SEQ ID NO: 3 72.5 72.7P091 76.5 77 P136 76.9 76.5 P155 76 76.3 P097 77.7 77.4 P140 78.1 77.5P161 79.2 78.5 P172 78.1 77.8 P173 78.5 77.6 P190 79.2 78.5

Example 10: Temperature Activity Measurement

Activity measurement of pullulanases was carried out in the range of55-77.5° C. at pH 5.0 by PHADEBAS assay described in EXAMPLE 2. Thetemperature optimum of the variants were higher than the parental P008,around 70-72.5° C. as shown in the below table.

Temperature (° C.) 55 60 65 67.5 70 72.5 75 77.5 SEQ ID 53% 79% 89%100%  93% 15%  2% 1% NO: 3 P161 25% 46% 99% 91% 98% 100%  20% 2% P17238% 56% 68% 90% 100%  85% 17% 1% P173 35% 55% 81% 97% 100%  89% 15% 1%P190 23% 36% 62% 88% 98% 100%  34% 2%

Example 11: Catalytic Domain Variants Having N-Terminal Parts Replaced

Two variants having substitutions in the catalytic domain, P240 andP265, described in example 3 were tested after replacement of theN-terminal domains. The replacement resulted in P256 and P267.

Bacillus clones were fermented in 96 well MTPs containing TB-gly medium(13.3 g/L Bacto™ Tryptone, 26.6 g/L Bacto™ Yeast extract D, 4.4 g/LGlycerol) with 6 mg/L chloramphenicol at 220 rpm, 37° C. and pullulanaseactivities were measured at several temperatures by Phadebas assaydescribed in EXAMPLE 2. Relative activity of pullulanase variantsshowing higher thermoactivity compared to parental pullulanases as shownin below table.

Relative activity at indicated temp (° C.) 65/60 67/60 70/60 72/60 SEQID NO: 19 88% 50% 9% 9% P256 105% 90% 17% 9% P267 96% 101% 28% 10%

Example 12: Relative Activity of Selected Variants

Purified enzyme was diluted to a fixed concentration with dilutionbuffer. Ten microliters of enzyme solution was added to 110 ul ofpreincubated 0.5% substrate solution (at 60-75° C. at 5° C. intervals)and incubated for 30 min. The reaction was stopped by adding 10 ul of500 mM NaOH and 40 ul of PAHBAH solution was added there. After 20 minincubation at 55° C., the absorbance at A405 was read.

Relative activity 65° C./60° C. 70° C./60° C. 70° C./75° C. Commercial65% 0% 0% PromozymeD P008 112% 117% 28% P199 122% 141% 119% P202 123%141% 125% P216 123% 137% 75% P219 118% 121% 62% P240 114% 58% 3% P252132% 136% 22% P254 115% 21% 6% P256 112% 40% 6% P303 125% 135% 68%

Relative activity 72° C./60° C. 74° C./60° C. P303 37% 12% P306 82% 29%P219 65% 20% Commercial PromozymeD 0% 0%

Example 13: Improved Specific Activity for Selected Variants

Specific activities toward Pindex100 (DE3) were determined using themethod described in a modified method of PAHBAH-Pullulan assay describedin EXAMPLE 2 using purified pullulanase samples (2 μg/ml) at 60° C. and65° C. Instead of using pullulan, Pindex100 was used in this experiment.The specific activities are listed as relative activity to that ofpurified commercial product, Promozyme® D2 (Sigma E2412)

Specific activity toward maltodextrin (100% at promozyme D at 60° C.)60° C. 65° C. Commercial PromozymeD2 100% 65% P199 108% 132% P202 110%135% P219 127% 150% P240 141% 161% P252 108% 143% P254 122% 140% P256128% 143% P303 110% 137% P380 97% 116%

Example 14: Relative Activity Measurements of Selected PullulanaseVariants

Relative activity measurements of selected pullulanase variants wascarried out in the range of 65-79° C. at pH 5.0 by the PHADEBAS assaydescribed in EXAMPLE 2. The results are shown in the tables below.

Relative activity 72° C./65° C. 74° C./65° C. 76° C./65° C. 78° C./65°C. P303 75% 12% 1% 0% P316 91% 36% P317 97% 42% P318 91% 39% P319 93%40% P323 86% 27% P324 74% 17% 2% P329 76% 21% P347 75% 13% P368 88% 62%P369 92% 59% 2% P370 105% 81% 11% P372 109% 87% 10% P375 113% 93% 4%P376 112% 98% 4% P377 93% 78% 16% P378 108% 86% P385 104% 63% 5% P39790% 46% 3% P415 46% 6% P416 32% P417 52% 8% P418 48% P424 60% 12% P42549% 7% P426 25% 3% P427 67% 20% P428 61% 14% P435 4% 2% P436 15% 4% P47247% 27% P484 45% 24% P485 80% 60% P486 79% 68%

Relative activity 72° C./65° C. 74° C./65° C. 76° C./65° C. 78° C./65°C. P202 109% 96% 26% 2% P287 107% 96% 42% 3% P380 101% 97% 78% 7%

Relative activity 76° C./65° C. 78° C./65° C. 79° C./65° C. P202 26% 2%P380 32% 7% P388 68% P398 51% 19% P403 70% 46% P408 92% 57% P423 87% 73%P430 90% 79% P431 93% 95% P443 100% 62% P444 77% 71% P448 81% 60% P44970% 60% P450 73% P470 84% 68% P481 89% 63% P482 78% 59% P483 62% 35%P493 87% 71% 33% P507 88% 74% 38%

Example 15: Enzyme Thermostability Measurement (TSA)

The thermo-stability measured as melting temperature (Tm) was determinedas described in example 9.

Purified enzyme was diluted with 50 mM sodium acetate pH 5.0 or 4.3 to0.5 mg/ml and mixed with the equal volume of SYPRO Orange (Invitrogen)diluted with Milli-Q water. Thirty microliters of mixture solution wastransfer to LightCycler 480 Multiwell Plate 96 (Roche Diagnostics) andthe plate was sealed.

Equipment parameters of TSA:

-   -   Apparatus: LightCycler 480 Real-Time PCR System (Roche Applied        Science)    -   Scan rate: 0.02° C./sec    -   Scan range: 37-96° C.    -   Scan rate: 1.26° C./min    -   Integration time: 0.5 sec    -   Excitation wave length 465 nm    -   Emission wave length 580 nm

The obtained fluorescence signal was normalized into a range of 0 and 1.The Melting temperature (Tm) was defined as the temperature where thenormalized value is closest to 0.5. Tm analysis by TSA

Tm (° C.) pH 4.3 pH 5.0 SEQ ID NO: 9 69 68 P202 75.7 76.07 P199 75.0575.5 P213 78.55 78.7 P225 80.29 80.74 P216 72.68 73.49 P217 73.08 74.1P218 72.05 72.65 P219 72.11 72.89 P252 71.53 72.08 P303 72.58 73.54 P38075.5 76.7 P370 74.1 74.6 P379 72.2 73.2 P368 73.4 73.9 P385 73.5 73.3P408 77.2 78.5 P423 71.6 74.7 P425 71.7 72.7 P470 80.1 80.3 P472 76.275.7

Example 16: Saccharification Test of Selected Variants

Maltodextrin which dextrose equivalent (DE) was adjusted to 11 wasprepared from a conventional starch liquefaction process using cornstarch and spray-dried for this experiment. The maltodextrin powder wasdissolved in hot milliQ water and the pH was adjusted by HCl/NaOH to be4.3 at 65° C., and then the solid was adjusted to 40% dry solid (DS) bymeasuring refractive index (RI) of the syrup. The syrup was conducted in25 ml glass vial, and saccharification was started at 65° C. by adding 2ml enzyme mixture containing purified glucoamylase JGA098 andpullulanase so that the final dosage of glucoamylase was 0.194 AGU/gDSand that of pullulanase was 5.3 or 10.7 ug/gDS. The samples wereincubated at 65° C. with stirring and were sampled at different timepoints. At each sampling, 0.75 ml of the syrup was heat-inactivated at100° C. for 15 min, and then diluted 6-fold with distilled water andfiltered through 0.2 pm nylon syringe filter prior to HPLC analysis.Twenty microliter of the syrup sample was applied to HPX-87C column(Bio-Rad) equilibrated with distilled water at flow rate of 0.5 ml/minat 85° C., and glucose and other oligosaccharides were fractionatedunder isocratic conditions and detected by a RI detector. Glucose yield(% DX) was calculated as the percentage of glucose peak area in totalarea detected. The enzyme dosages and the % DX against incubation timewere shown in the Table below.

% DX of the syrup at different time points. Time PromozymeD2 P256 P240PromozymeD2 P256 P240 [hr] 5.3 ug/gDS 5.3 ug/gDS 5.3 ug/gDS 10.7 ug/gDS10.7 ug/gDS 10.7 ug/gDS 8 62.7 62.4 62.8 63.6 63.3 63.3 16 79.7 80.881.1 83.1 83.0 83.8 24 86.7 87.8 88.8 90.5 90.7 91.3 36 91.3 92.5 93.394.5 94.9 95.2 48 93.5 94.5 95.1 95.8 96.1 95.9 60 94.5 95.4 95.7 96.096.3 96.2 72 95.0 95.6 95.8 96.1 96.2 96.2 90 95.2 95.7 95.7 95.9 96.196.0

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

1. A pullulanase variant comprising a substitution at one or morepositions corresponding to positions 393, 143, 150, 243, 244, 346, 368,370, 373, 381, 385, 387, 402, 429, 430, 456, 486, 492, 610, 631, 632,665 and 699 of the polypeptide of SEQ ID NO: 3 wherein the variant haspullulanase activity, and; a) wherein the variant has at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 3 and wherein the variant has increased thermoactivitycompared to the pullulanase of SEQ ID NO: 3, particularly the varianthas at least 60% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 65%, more particularly atleast 70%, more particularly at least 60%, more particularly at least75%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%; or b) wherein the variant has at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 6 and wherein the variant has increasedthermoactivity compared to the pullulanase of SEQ ID NO: 6, particularlythe variant has at least 30% relative activity when measured at 70° C.relative to activity at 65° C., more particularly at least 40%, moreparticularly at least 50%, more particularly at least 60%, moreparticularly at least 70%, more particularly at least 80%, moreparticularly at least 90%, more particularly at least 100%; or c)wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 9 and whereinthe variant has increased thermoactivity compared to the pullulanase ofSEQ ID NO: 9, particularly the variant has at least 30% relativeactivity when measured at 70° C. relative to activity at 65° C., moreparticularly at least 40%, more particularly at least 50%, moreparticularly at least 60%, more particularly at least 70%, moreparticularly at least 80%, more particularly at least 90%, moreparticularly at least 100%; or d) wherein the variant has at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 16 and wherein the variant has increased thermoactivitycompared to the pullulanase of SEQ ID NO: 16, particularly the varianthas at least 30% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 40%, more particularly atleast 50%, more particularly at least 60%, more particularly at least70%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%; or e) wherein the variant has at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 17 and wherein the variant has increasedthermoactivity compared to the pullulanase of SEQ ID NO: 17,particularly the variant has at least 30% relative activity whenmeasured at 70° C. relative to activity at 65° C., more particularly atleast 40%, more particularly at least 50%, more particularly at least60%, more particularly at least 70%, more particularly at least 80%,more particularly at least 90%, more particularly at least 100%.
 2. Thevariant according to claim 1 wherein the variant comprises or consistsof one or more substitutions selected from the group consisting of 393A,143G, 150R, 243E, 244K, 345P, 346S, 368G, 370S, 373L, 381V, 382T, 385F,387L, 402T, 429V, 430R, 431E, 432F, 456A, 486C, 492S,A, 610R,L, 624S,631S, 632C, 665I and 699R.
 3. The variant of claim 1, wherein thevariant comprises at least one of the following substitutions orcombinations of substitutions: N368G; E699R; E150R; N346S; N243E; S244K;V143G; N393A; N610R; N610L; G624S; F456A; T492S,A; V486C+T492S,A;N368G+M402T; T631S+S632C; V486C+T492S,A+T631S+S632C; N393A+T631S+S632C;T631S+S632C+E699R; N393A+V486C+T492S,A+T631S+S632C; N393A+G624S+S632C;N393A+N610R+T631S+S632C; N393A+G624S+T631S+S632C;N393A+N610R+G624S+T631S+S632C; N393A+V486C+T492S,A+G624S+T631S+S632C;N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+V486C+T492S,A+N610R+G624S+T631S+S632C+E699R;N346S+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+F456A+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;A345P+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;N368G+K370S+I373L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;I381V+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;I381V+N382T+Q385E+Q387L+N393A+V486C+T492S,A+N610R+G624S+T631S+S632C;A345P+N368G+N393A+T492S,A+N610R+G624S+T631S+S632C;N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C;A345P+N368G+I381V+Q385E+Q387L+N393A+T492S,A+N610R+G624S+T631S+S632C+V665I;N393A+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+I429V+Q431E+V486C+T492S,A+N610R+G624S+T631S+S632C;N393A+I429V+T430R+Q431E+L432F+V486C+T492S,A+N610R+G624S+T631S+S632C;N368G+N393A+A492S,A; N368G+N393A; N393A+N610R; N368G+N393A+N610R;N368G+N393A+T492S,A+N610R+G624S; N368G+N393A+T492S,A+N610R+G624S+T631S+S632C+Q431E+L432F; N368G+N393A+N610R+G624S+T631 S+S632C;N368G+N393A+T492S,A+N610R+G624S+T631S+S632C N368G+N393A+N610R+G624S+T631S+S632C+Q431E+L432F.
 4. A variant catalytic domain, wherein the variantcatalytic domain comprises a substitution at one or more positionscorresponding to positions 393, 346, 368, 370, 373, 381, 385, 387, 402,429, 430, 456, 486, 492, 531, 532, 610, 631, 632, 665 and 699 of thepolypeptide of SEQ ID NO: 3; and; a) wherein the variant catalyticdomain has at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%, but less than 100% sequenceidentity to amino acids 330 to 828 of SEQ ID NO: 3; or b) wherein thevariant catalytic domain has at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to amino acids 430 to 928 of SEQ ID NO: 6; or c)wherein the variant catalytic domain has at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to amino acids 330 to 829 of SEQ IDNO: 9; or d) wherein the variant catalytic domain has at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%, but less than 100% sequence identity to amino acids 430 to928 of SEQ ID NO: 16; or e) wherein the variant catalytic domain has atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity toamino acids 430 to 928 of SEQ ID NO:
 17. 5. A pullulanase variantcomprising the variant catalytic domain according to claim 10, whereinthe pullulanase variant has pullulanase activity and increasedthermoactivity compared to the parent pullulanase, and the variant hasat least 60% relative activity when measured at 70° C. relative toactivity at 65° C., more particularly at least 65%, more particularly atleast 70%, more particularly at least 60%, more particularly at least75%, more particularly at least 80%, more particularly at least 90%,more particularly at least 100%.
 6. The pullulanase variant according toclaim 5, wherein the variant comprises or consists of one or moresubstitutions selected from the group consisting of 393A, 345P, 346S,368G, 370S, 373L, 381V, 382T, 385F, 387L, 402T, 429V, 430R, 431E, 432F,456A, 486C, 492S,A, 610R,L, 624S, 631S, 632C, 665I and 699R.
 7. Thepullulanase variant according to claim 6, further comprising anN-terminal part comprising at least one of the domains selected from aCBM41 domain, an X45 domain and a CBM48 domain.
 8. The pullulanasevariant according to claim 7, comprising an N-terminal part comprising aCBM41 domain, an X45 domain and a CBM48 domain.
 9. The pullulanasevariant according to claim 8, further comprising an X25 domain.
 10. Amethod for producing a variant pullulanase of a parent pullulanasecomprising substitution of the parent pullulanase at one or morepositions corresponding to positions 393, 143, 150, 243, 244, 345, 346,368, 370, 373, 381, 382, 385, 387, 402, 429, 430, 431, 432, 456, 486,492, 531, 532, 610, 624, 631, 632, 665 and 699 of the polypeptide of SEQID NO: 3 wherein the variant has pullulanase activity and increasedthermoactivity compared to the parent; and; a) wherein the variant hasat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99%, but less than 100% sequence identity to thepolypeptide of SEQ ID NO: 3; or b) wherein the variant has at least 85%,at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, orat least 99%, but less than 100% sequence identity to the polypeptide ofSEQ ID NO: 6; or c) wherein the variant has at least 85%, at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%,but less than 100% sequence identity to the polypeptide of SEQ ID NO: 9;or d) wherein the variant has at least 85%, at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO: 16; or e)wherein the variant has at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100% sequence identity to the polypeptide of SEQ ID NO:
 17. 11. Avariant pullulanase produced by the method of claim
 10. 12. Acomposition comprising the variant pullulanase of claim
 1. 13. Thecomposition according to claim 12, further comprising one or moreenzymes selected from the group consisting of: glucoamylase,alpha-amylase, beta-amylase, and protease.
 14. The composition accordingto claim 12, comprising a variant pullulanase of claim 1 and: i) aglucoamylase, an alpha-amylase and a protease; ii) an alpha-amylase anda protease; iii) a glucoamylase and an alpha-amylase; iv) abeta-amylase; or v) a glucoamylase.
 15. A process of producing afermentation product from starch-containing material comprising thesteps of: (a) liquefying starch-containing material in the presence ofan alpha amylase; (b) saccharifying the liquefied material in thepresence of a glucoamylase; and (c) fermenting with a fermentingorganism; wherein step (a) and/or step (b) is carried out in thepresence of a variant pullulanase of claim
 1. 16. A process of producinga fermentation product from starch-containing material, comprising thesteps of: (a) saccharifying starch-containing material at a temperaturebelow the initial gelatinization temperature of said starch-containingmaterial; and (b) fermenting with a fermenting organism wherein step (a)is carried out using at least a glucoamylase, and a variant pullulanaseof claim
 1. 17. A polynucleotide encoding the variant pullulanase ofclaim
 1. 18. A nucleic acid construct or expression vector comprisingthe polynucleotide of claim
 17. 19. A host cell comprising thepolynucleotide of claim
 17. 20. A whole broth formulation or cellculture composition comprising a variant pullulanase of claim 1.